CN104403944B

CN104403944B - The automation equipment cultivated for ovary cell vitro fertilization and the spilting of an egg and method

Info

Publication number: CN104403944B
Application number: CN201410626035.6A
Authority: CN
Inventors: 徐小杨
Original assignee: Guangzhou Lanri Biotechnology Co ltd
Current assignee: Guangzhou Lanri Biotechnology Co ltd
Priority date: 2014-11-06
Filing date: 2014-11-06
Publication date: 2016-11-30
Anticipated expiration: 2034-11-06
Also published as: CN104403944A

Abstract

The present invention provides a kind of automation equipment cultivated for ovary cell vitro fertilization and the spilting of an egg, and the central controller and the ovum that including automatic communication type partition conbination incubator, are placed in outside incubator identify sorting unit automatically；Incubator is provided with the operator scheme device of different phase in different subregions, and each subregion realizes connecting/separating by automatic gas-tight door；It is provided with the culture carrier for carrying ovum drop in incubator and drives culture carrier to travel to and fro between the culture carrier driving means of each subregion；Ovum identifies the sorting unit control instruction according to reception automatically, after the follicle stock solution with ovum is identified and is sorted, on the culture carrier in output ovum drop to incubator；When automatic gas-tight door is opened according to control instruction, culture carrier driving means drives described culture carrier to travel to and fro between each subregion according to control instruction, and ovum drop enters each subregion to carry out the operator scheme of correspondence.

Description

Automatic device and method for egg cell in-vitro fertilization and cleavage culture

Technical Field

The invention relates to a cell culture device, in particular to an automatic device and method for in-vitro fertilization and cleavage culture of egg cells.

Background

The artificial assisted reproduction technology is developed and applied to clinic at the end of the 20 th century, is popularized and popularized worldwide at the beginning of the 21 st century, and the technology developers gain the recognition of all mankind. The technology is popularized worldwide, and will continue to bring benefits to all mankind.

The artificial assisted reproduction is the combination of engineering technology and clinical medicine technology. The development of clinical medicine solves the problem of germ cell source (mainly human oocyte source in a specific development stage and confirmed sperm in-vitro insemination capability), and defines the physiological elements of pregnancy; the engineering technology solves the problems of in vitro fertilization and simulation of in vivo environment in the process of in vitro culture of fertilized eggs. The existing general IVF operation method mainly comprises the manual operation of an open environment outside an incubator, the outside operation is separated from the culture environment, the method depends on a buffer system, the temperature control device outside the culture environment, the operation technology and responsibility of an operator are highly dependent, the manual operation is required, the operation environment is maintained depending on specific operation rules and equipment, the working process is not traceable and can not be standardized.

The procedure for IVF laboratory serial culture is briefly described as follows (taking the transplantation mode on day 3 as an example): preparing a culture solution in advance according to a surgical plan; the operation aspirates the follicular fluid, the operation assistant instructs the operator whether the follicular fluid container (test tube) needs to be replaced, and the operator continues to aspirate or stops aspirating according to the prompt; after the follicular fluid is filled into the test tube, the surgical assistant puts into the constant temperature test tube rack, replaces a new test tube container, and repeats the above process until the surgical person finishes suction to complete the operation; taking out the follicular fluid containing the cumulus oophorus compound by a laboratory operator, completely transferring the follicular fluid into an ovum picking container, quickly identifying the cumulus oophorus cell compound under the assistance of a hypoploid visual microscope, manually transferring the cumulus oophorus cell compound into buffer fluid (the buffer fluid container is placed on a constant temperature platform), manually washing the cumulus oophorus cell compound, transferring the cumulus oophorus cell compound into insemination culture fluid (the insemination culture fluid container is placed on the constant temperature platform), transferring the insemination culture fluid into an incubator environment, adding sperm liquid drops (outside the incubator) at a specified time, manually removing granular cells (outside the incubator) at a specified time after completing insemination in the incubator, quickly transferring fertilized eggs to an ovum lysis culture fluid outside the incubator, completing transplantation or freeze-thaw preservation at the specified time, and also continuously culturing the fertilized eggs to be transplanted or; if the operator decides to perform intracytoplasmic sperm microinjection insemination, the laboratory operator must make a buffer solution droplet system, prepare an insemination culture dish, complete microinjection insemination under an inverted microscope with a constant temperature platform device, then manually transfer the inseminated cells into the culture dish (insemination culture droplet), place into a culture box, and continue culturing.

In the working system, culture liquid drops are transferred for multiple times in an open environment and an artificial environment in an incubator, target cells are also transferred for multiple times in different liquid environments, the cell transfer is manually completed by an operator (a hand-held suction pipe tool is used for manually operating single cells, operation errors such as bubble generation and the like are strictly avoided), the culture environment is discontinuous, and the cells are easily lost and damaged; in the working system, the cell culture environment cannot be standardized, the operation processes of any two groups of cells cannot be strictly compared, the operation processes cannot be monitored, and whether the operation is successful can be judged only from the transplantation result.

Under the current working rules, the success rate of embryo culture and clinical transplantation in clinical reproduction laboratories is directly related to the proficiency and responsibility of embryo operators and the management behaviors of laboratories, which is an important reason that the final planting success rates of various clinical reproduction medical units and operators show obvious differences. Qualified laboratory operators require strict operational training and long-term, large amounts of practice, which limits the further popularization of IVF technology and causes a great deal of waste of social resources.

Disclosure of Invention

The invention aims to provide an automatic device for in-vitro fertilization and cleavage culture of egg cells, which simulates the environment of human egg cell fertilization and the early development process of fertilized eggs and can realize automatic acquisition of cumulus complexes, addition of sperm suspension into culture liquid drops, degranulation of the cumulus complexes, automatic control of the components of the culture liquid drops, full-process controlled culture of blastocysts and complete transfer of the culture liquid drops containing embryos without residues.

The embodiment of the invention provides an automatic device for in-vitro fertilization and cleavage culture of egg cells,

comprises an automatic communication type subarea combined incubator, a central controller arranged outside the incubator and an automatic egg cell identification and sorting device;

the automatic communication type subarea combined incubator is provided with operation mode devices at different stages in the process of fertilization of egg cell liquid drops and culture of cleavage in different subareas, and each subarea is communicated/separated through an automatic airtight door; the automatic communication type partitioned combined incubator is internally provided with a culture carrier for bearing egg cell liquid drops and a culture carrier driving device for driving the culture carrier to move to and fro each partition;

the automatic egg cell identification and sorting device, the operation mode devices at different stages, the automatic airtight door and the culture carrier driving device are all connected with the central controller to receive corresponding control instructions;

the automatic egg cell identification and sorting device identifies and sorts follicle stock solution with egg cells according to the received control instruction, and then outputs egg cell liquid drops to a culture carrier in the automatic communication type partition combined culture box; when the automatic airtight door is opened according to a control instruction, the culture carrier driving device drives the culture carrier to move back and forth between each subarea of the automatic communication type subarea combined culture according to the control instruction, so that the egg cell liquid drops on the culture carrier enter each subarea to carry out a corresponding operation mode.

As an improvement of the technical scheme, the automatic communicated type partition combined incubator comprises two partitions, and the two partitions are composed of a main incubator body, an auxiliary incubator body and the automatic airtight door used for realizing communication/separation between the main incubator body and the auxiliary incubator body.

As an improvement of the above solution, the operation mode devices include, but are not limited to, direct insemination mode devices, vitrification freeze/resuscitation mode devices, and transplantation mode devices.

As an improvement of the technical proposal, the direct insemination mode device is arranged in the main incubator; the vitrification freezing/resuscitation mode device and the transplantation mode device are arranged in the auxiliary incubator body.

As an improvement of the above technical solution, the direct insemination mode device comprises a first liquid drop changing operation device, the first liquid drop changing operation device comprises a waste liquid extraction controller, a culture solution supplement controller, a liquid drop output pipeline and a liquid drop input pipeline, and two terminals of the liquid drop output pipeline and the liquid drop input pipeline are connected with liquid drop operation needles to suck/output liquid drops; the waste liquid extraction controller extracts waste liquid in cell drops on the culture carrier through the drop output pipeline and controls the flow of the extracted waste liquid, and the culture liquid supplement controller conveys culture liquid to the cell drops on the culture carrier through the drop input pipeline and controls the flow of the conveyed culture liquid; and the central controller is respectively connected with the waste liquid extraction controller and the culture solution supplement controller so as to control the waste liquid extraction controller and the culture solution supplement controller to work.

As an improvement of the technical scheme, the waste liquid extraction controller and the culture solution supplement controller are peristaltic pumps or stepping motors.

As an improvement of the above technical solution, the direct insemination mode apparatus further comprises a cell image acquisition device connected with the central controller, for acquiring cell image information in the cell droplets on the culture carrier when operating in direct insemination mode.

As an improvement of the technical scheme, a plurality of pits for bearing the egg cell liquid drops are arranged on the surface of the culture carrier, and the surfaces of the pits are super-hydrophobic surfaces.

As an improvement of the technical proposal, a closed annular limiting part is convexly arranged on the edge of each pit on the surface of the culture carrier, and the surface of the limiting part is an oleophilic surface.

As an improvement of the above technical solution, the automatic egg cell identification and sorting device includes a negative pressure generator, an egg cell image acquisition device, a light source device, a follicular fluid collection container and a sorting switch, and the central controller is connected with the negative pressure generator, the egg cell image acquisition device and the sorting switch respectively;

the sorting switch comprises a first valve port, a second valve port and a third valve port, wherein the first valve port is connected between a main input conduit for inputting follicle stock solution with ova and a main output conduit for outputting the ova to a culture carrier, the second valve port is connected between the main input conduit and a secondary output conduit for outputting follicle solution without the ova to the follicular fluid collecting container, and the third valve port is connected between a secondary input conduit for inputting specific culture solution and the main output conduit; the negative pressure generator is communicated with the follicular fluid collecting container through a communicating conduit;

the light source equipment is arranged on one side outside the main input catheter, illuminates the inside of the main input catheter and images on the egg cell image acquisition equipment arranged on the other side outside the main input catheter; when the negative pressure generator is started, the follicle stock solution with the ova flows into the main input conduit, and when the follicle stock solution flows through the oocyte image acquisition device, the oocyte image acquisition device acquires oocyte image information, the central controller controls the opening/closing of the first valve port, the second valve port and the third valve port of the sorting switch according to the oocyte image information, so that the ova in the follicle stock solution with the ova flow out from the first valve port, the follicle fluid without the ova flows out from the second valve port and flows into the follicle fluid collection container, and the ova flowing out from the first valve port and the specific culture fluid flowing in from the third valve port form an oocyte drop and then flow out to the culture carrier.

As an improvement of the above technical solution, the central controller controls the operation of the negative pressure generator to control the flow rate of the follicular fluid with ova in the main input conduit.

As an improvement of the above technical solution, a duct length difference between the main input duct position corresponding to the light source device and the egg cell image acquisition device and the main input duct position at which the first valve port is located is a preset value.

As an improvement of the above technical solution, the central controller calculates, according to the egg cell image information, and by combining the flow rate of the follicle stock solution with egg cells in the main input conduit and the conduit length difference, the on/off time of the first valve port, the second valve port, and the third valve port of the sorting switch.

As an improvement of the technical proposal, the vitrification freezing/recovery mode device comprises a carrier mechanical action device, a second liquid drop changing device, a freezing/recovery carrier, an operation platform for bearing the freezing/recovery carrier and a freezing medium container, wherein,

the carrier mechanical action device and the second liquid drop changing device are respectively connected with the central controller to receive corresponding freezing/resuscitation control commands;

the carrier mechanical action device comprises a mechanical body, and a carrier clamping action device and a liquid drop pickup device which are arranged on the mechanical body; the liquid drop pickup device is used for correspondingly adsorbing and transferring target cell liquid drops to a specific position of the freezing/resuscitation carrier according to the freezing/resuscitation control instruction; the carrier clamping action device is used for moving the target cell drops after the liquid drop changing operation on the freezing/thawing carrier and the freezing/thawing carrier into the freezing medium container together according to a freezing/thawing control instruction so as to complete freezing/moving the target cell drops after freezing in the freezing medium container and the freezing/thawing carrier from the freezing medium container to the operation table;

the second liquid drop changing device is used for carrying out liquid drop changing operation on target cell liquid drops on the freezing/resuscitation carrier carried by the operation platform according to the freezing/resuscitation control instruction.

As an improvement of the above technical solution, the second droplet changing device and the first droplet changing device have the same structure.

As an improvement of the above technical solution, the vitrification freezing/resuscitation mode device further comprises an image acquisition device connected with the central controller; the image acquisition device is used for acquiring cell image information in the cell liquid drops on the freezing/resuscitation carrier and sending the acquired cell image information to the central controller for processing.

As an improvement of the technical scheme, the carrier body of the freezing/resuscitation carrier is of a sheet structure with an arc-shaped inner surface, a super-hydrophobic surface functional area and a hydrophilic surface functional area are arranged on the inner surface, the periphery of the hydrophilic surface functional area is surrounded by the super-hydrophobic surface functional area, and a liquid drop positioning mark is arranged on the hydrophilic surface functional area.

As an improvement of the above technical solution, the superhydrophobic surface functional region is a circular ring-shaped region, and the hydrophilic surface functional region is a circular region.

As an improvement of the above technical solution, a center point of the hydrophilic surface functional region coincides with a lowest point of the carrier body.

As an improvement of the above technical solution, the transplantation mode device can realize transplantation function by the vitrification freezing/resuscitation mode device.

As an improvement of the technical scheme, the main culture box body/the auxiliary culture box body are internally and respectively provided with an environment management device for controlling and adjusting the temperature, the humidity, the pressure and the gas partial pressure in the main culture box body/the auxiliary culture box body.

As an improvement of the technical proposal, the main culture box body/the auxiliary culture box body is provided with a flow path communicated with the outside, and cell liquid drops to be cultured sent in from the outside are received through the flow path or cell liquid drops after culture are sent out to the outside.

The embodiment of the invention also provides an automatic method for the in vitro fertilization and the cleavage culture of the egg cells, and the automatic device is used for carrying out the in vitro fertilization and the cleavage culture on the egg cells, and the operation modes comprising a cumulus complex identification and sorting mode, a direct insemination mode, a vitrification freezing/reviving mode and a transfer mode are all used for operating the egg cell liquid drops.

Compared with the prior art, the automatic device for in-vitro fertilization and cleavage culture of the egg cells disclosed by the invention simulates the environment of the process of fertilization of the human egg cells and early development of fertilized eggs, and can realize automatic acquisition of a cumulus complex, addition of sperm suspension into culture liquid drops, degranulation of the cumulus complex, automatic control of the components of the culture liquid drops, full-process controlled culture of blastocysts and complete transfer of the culture liquid drops containing embryos without residues. Need not artificial intervention culture environment under normal condition, apparent culture process whole record, the parameter of culture environment also can whole record, apparent culture process can be traced back completely, can absorb automatically after embryo culture accomplishes to automatic tubulation according to the transplantation requirement hands over the person, also can carry out automatic cryopreservation operation or carry out automatic recovery operation to freezing and thawing embryo to the target cell, accomplish work flow.

Drawings

FIG. 1 is a block diagram of an automated apparatus for in vitro fertilization and cleavage culture of egg cells according to an embodiment of the present invention.

FIG. 2 is a schematic view showing the structure of a living cell incubator according to an embodiment of the present invention.

Fig. 3 is a schematic structural diagram of an automatic egg cell identification and sorting device in an embodiment of the invention.

Fig. 4 is a block diagram showing the configuration of a sorting switch of the automatic egg cell sorting apparatus shown in fig. 3.

FIG. 5 is a schematic top view of the structure of a living cell culture carrier according to an embodiment of the present invention.

FIG. 6 is a schematic sectional view showing the structure of a living cell culture carrier according to an embodiment of the present invention.

FIG. 7 is an enlarged schematic view of a living cell culture carrier according to an embodiment of the present invention.

Fig. 8 is a schematic view of a direct insemination mode device in accordance with an embodiment of the present invention.

Fig. 9 is a block diagram of a vitrified freeze/resuscitation mode device in accordance with an embodiment of the present invention.

Fig. 10 is a schematic diagram of a freezing/resuscitation vehicle according to an embodiment of the present invention.

FIG. 11 is a schematic diagram of the configuration of the console of a vitrification freeze/resuscitation mode device in an embodiment of the present invention.

Fig. 12 is a schematic diagram of the structure of a carrier mechanical actuation device of a vitrified freeze/resuscitation mode device in an embodiment of the invention.

FIG. 13 is a schematic diagram of a second droplet changing apparatus of a vitrified freeze/resuscitate mode apparatus in accordance with an embodiment of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Referring to fig. 1, an automatic apparatus for in vitro fertilization and cleavage culture of egg cells according to an embodiment of the present invention includes an automatic communication type partitioned combined incubator 100, a central controller 9 disposed outside the incubator, and an automatic egg cell identification and sorting apparatus 200; wherein:

the automatic communicating type partitioned combined incubator 100 includes a main incubator body 101, a sub-incubator body 102, and an automatic airtight door 103 for achieving communication/separation between the main incubator body 101 and the sub-incubator body 102. A culture carrier 1 for bearing the liquid drops of the egg cells is arranged in the main culture box body 101/the auxiliary culture box body 102; and operation mode devices used for performing fertilization on the egg cell liquid drops and different stages in the process of culturing the cleavage are respectively arranged in the main culture box body/the auxiliary culture box body. A culture carrier driving device 105 for driving the culture carrier 1 to reciprocate between the main culture box 101 and the auxiliary culture box 102 when the automatic airtight door 103 is opened is arranged inside/outside the main culture box 101/the auxiliary culture box 102;

the egg cell automatic identification and sorting device 200, the operation mode devices 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 instructions;

the automatic egg cell identification and sorting device 200 identifies and sorts the follicle stock solution with egg cells (cumulus cell complex) according to the received control instruction, and then outputs egg cell liquid drops to the culture carrier in the automatic communication type partition combination incubator 100; when the automatic airtight door is opened according to a control instruction, the culture carrier driving device 105 drives the culture carrier 1 to move back and forth between the main culture box 101 and the auxiliary culture box 102 according to the control instruction, so that the egg cell liquid drops on the culture carrier 1 enter the main culture box/the auxiliary culture box to perform a corresponding operation mode.

The detailed structure and operation principle of each component of the automated device for in vitro fertilization and cleavage culture of egg cells according to the embodiment of the present invention will be described in detail with reference to fig. 2 to 13.

First, referring to FIG. 2, the automated linked type partitioned incubator 100 includes a main culture incubator body 101 and a sub-culture incubator body 102, and the main culture incubator body 101 and the sub-culture incubator body 102 are communicated/partitioned with each other by providing an automated airtight door 103. The living cell incubator 100 further comprises an automatic airtight door driving device 108, and the automatic airtight door driving device 108 controls the opening/closing of the automatic airtight door 103. The automatic airtight door driving device 108 may be provided inside or outside the incubator body 101/sub-incubator body 102, and may control the opening/closing of the automatic airtight door 103 by contact/non-contact with the automatic airtight door 103.

A culture carrier 1 for carrying the liquid drop of the egg cell is arranged in the main culture box body 101/the auxiliary culture box body 102. The automatic communication type partitioned combined incubator 100 is further provided with an incubation carrier driving device 105 for driving the incubation carrier 1 to reciprocate between the main incubator body 101 and the sub-incubator body 102 when the automatic airtight door 103 is opened. The culture carrier driving device 105 may be disposed inside or outside the main culture box 101/sub culture box 102, and may drive the culture carrier 1 to move by contacting/not contacting the culture carrier 1.

Operation mode devices for fertilizing the egg cell drop (carried on the culture carrier 1) and performing different stages of the culture process are respectively arranged in the main culture box 101/the auxiliary culture box 102. The operational mode devices include, but are not limited to, direct insemination mode devices 300, freeze/resuscitation mode devices 400, and transplant mode devices (not shown), among others. For example, direct insemination mode apparatus 300 is provided inside main culture housing 101, and freezing mode apparatus, freeze-thaw cell recovery mode apparatus, and transplantation mode apparatus are provided inside sub culture housing 102. When the egg cell drop obtained by the cumulus cell compound sorting device 200 in the external open environment enters the sub-culture box 102 (carried on the culture carrier 1), the culture carrier 1 is moved from the sub-culture box 102 to the inside of the main culture box 101 by controlling the automatic airtight door 103 to open, and the fertilization mode operation is performed on the egg cell drop on the culture carrier 1 by the direct insemination mode device 300 in the inside of the main culture box 101. After completion of the fertilization mode operation, if a transplant mode, a freezing mode or a freeze-thaw cell recovery mode is required, the central controller 9 controls the automatic air-tight door 103 to open, moves the culture carrier 1 from the main culture chamber 101 to the inside of the sub-culture chamber 102, and performs a corresponding mode operation by a transplant mode device or a freezing/recovery mode device inside the sub-culture chamber 102.

Further, main culture casing 101/sub-culture casing 102 is provided with a flow path 106 communicating with the outside (in the present embodiment, flow path 106 is shown as being provided in sub-culture casing 102, but it is understood that flow path 106 may be provided) and main culture casing 101 receives the egg to be cultured (liquid drop) fed from the outside through flow path 106 or sends the cultured egg drop to the outside. The automatic egg cell identification and sorting device 200 is arranged in the external environment of the automatic communication type partition combined incubator, the automatic egg cell identification and sorting device 200 is used for identifying and obtaining the cumulus cells, liquid drops containing the cumulus cells are sent to the culture carrier 1 in the auxiliary culture box body 102 through the flow path 106 after specific culture solution is added to the cumulus cells, and then the subsequent operations such as a direct insemination mode, a freezing mode, a freeze-thaw cell recovery mode, a transplantation mode and the like are carried out on the egg cell liquid drops on the culture carrier 1 through the internal devices of the main culture box body 101/the auxiliary culture box body 102.

In the main culture tank 101 and the subsidiary culture tank 102, there are provided environment control devices 122a and 122b for controlling and adjusting the temperature, humidity, pressure and partial pressure of gas in the main culture tank 101 and the subsidiary culture tank 102, respectively. The environment inside the main/subsidiary culture cases 101, 102 is monitored and controlled by the environment management means 122a, 122b, thereby ensuring stability and continuity of the respective modes of operation.

The culture carrier driving device 105, the automatic airtight door driving device 108, the environment management devices 122a and 122b, and the operation mode devices are all electrically connected to the central controller 9, so as to realize one-way/two-way communication, and send the acquired information to the central controller 9 for processing, and receive the control command of the central controller 9 for corresponding operation.

Preferably, the automated apparatus for in vitro fertilization and cleavage culture of egg cells according to the present embodiment further includes a display 10 connected to the central controller 9 outside the main culture housing 101/sub culture housing 102, and the central controller 109 processes the received image information of each mode state transmitted from each apparatus, for example, the image information of each marker position is sequentially stored as an image set of a specific position and transmitted to the display 10 to be displayed. The operator can read the image set at any marked position through the display 10 at any time, judge the development state of the egg cells and record the development state. In addition, the central controller 9 can be shared with a comprehensive information management system of a hospital, an operator can make a reference at any time, and a patient can receive the information through a public information platform.

It can be seen that the automatic communication type partitioned combined incubator 100 disclosed in this embodiment realizes communication/separation of the inside of the main culture incubator 101 and the sub-culture incubator 102 by providing the automatic airtight door 103, and the main culture incubator 101/the sub-culture incubator 102 are respectively provided with operation mode devices for performing fertilization and different stages of the culture process on the egg cell liquid drops, and then the culture carrier driving device 105 drives the culture carrier to move back and forth between the main culture incubator 101 and the sub-culture incubator 102 when the automatic airtight door is opened, so that the egg cell liquid drops on the culture carrier enter the main culture incubator/the sub-culture incubator to perform corresponding operation modes. Therefore, the whole culture process is in the incubator, and the cell culture environment between the main culture box body and the auxiliary culture box body is controlled to be continuous, so that the problems of cell loss, damage and the like can be effectively avoided. In addition, the cell culture environment of the main culture box body and the auxiliary culture box body is standardized, so that the whole operation process can be effectively monitored, and the successful operation is facilitated.

Referring to fig. 3 to 4, the present invention is a schematic structural diagram of an automatic egg cell identification and sorting apparatus 200 according to an embodiment of the present invention. The automatic egg cell identifying and sorting device 200 comprises 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 a central controller 9 which is respectively connected with the negative pressure generator 201, the egg cell image acquiring device 202 and the sorting switch 205, wherein the negative pressure generator 201, the follicular fluid collecting container 204 and a conduit for sucking and conveying the follicular fluid with the egg cells are communicated, and the negative pressure generator 201 is used for forming negative pressure on the conduit to complete puncture and suction of the follicular fluid with the egg cells and enter the conduit. The oocyte image obtaining device 202 and the light source device 203 are arranged on two sides of a conveying path of the catheter to cooperatively obtain oocyte image information and send the obtained oocyte image information to the central controller 9, and the central controller 9 controls the on/off of a sorting switch 205 arranged on the catheter according to the oocyte image information, so that the separation of the oocytes in the follicle stock solution with the oocytes and the follicular fluid is realized, and the required oocytes are obtained.

Specifically, as shown in fig. 3, the sort switch 205 includes a first valve port 205a, a second valve port 205b, and a third valve port 205c, wherein:

the first port 205a is connected between a main inlet line 210 for feeding the follicle stock solution 20 with the ova 21 and a main outlet line 211 for discharging the ova 21 to the culture carrier 1. That is, the main input conduit 210 and the main output conduit 211 are communicated/isolated by the opening/closing of the first valve port 205a of the sorting switch 205.

The second port 205b is connected between the main input pipe 210 and a secondary output pipe 212 for outputting the follicular fluid 22 without the ova 21 to the follicular fluid collection container 204. That is, the main input conduit 210 and the sub output conduit 212 are connected/isolated by opening/closing the second valve port 205b of the sorting switch 205.

The third valve port 205c is connected between the secondary inlet conduit 213 for inputting the specific culture solution 23 and the main outlet conduit 211; i.e. the communication/isolation between the secondary input conduit 213 and the primary output conduit 211 is achieved by the on/off of the sorting switch 205. The secondary inlet conduit 213 is provided with a micro-flow pump 207 for controlling the flow of the specific culture solution 23 pumped out and flowing into the third port 205 c. The micro flow pump 207 is connected to the central controller 9 to receive control commands.

As shown in fig. 4, in the present embodiment, the sorting switch 205 may be implemented by using a three-way valve including a valve body 2051 having the first port 205a, the second port 205b, and the third port 205c, and a valve controller 2052 controlling opening/closing of the first port 205a, the second port 205b, and the third port 205c, and the central controller 9 is connected to the valve controller 2052. The valve control device 2052 controls the opening/closing of the first valve port 205a, the second valve port 205b, and the third valve port 205c in turn according to a command sent from the central controller 9.

The negative pressure generator 201 communicates with the follicular fluid collection container 204 via a communication conduit 214, thereby communicating with the secondary output conduit 212, and further communicating with the main input conduit 210 when the second valve port 205b is opened. The central controller 9 controls the operation of the negative pressure generator 201 under the control of the opening of the second valve port 205b, so that the flow rate V of the follicular fluid 20 with the ova 21 in the main inlet conduit 210 can be controlled.

The light source device 203 is disposed on the conveying path of the main input catheter 210 and located at one side outside the main input catheter 210, and the egg cell image acquisition device 202 is disposed at the other side outside the main input catheter and faces the light source device 203. The light source device 20 illuminates the inside of the main input catheter 210 and images on the egg image acquisition device 202. Preferably, the light source device 203 is a fiber-optic cold light source device, and the egg cell image acquisition device 202 is a CCD. The cell image imaged by the CCD is sent to the central controller 9 for image processing to obtain a more complete image.

In this embodiment, the main input conduit 210 is a transparent conduit, and the diameter D of the main input conduit 210 is a fixed value. The initial end of the main input catheter 210 is connected with a puncture needle and other devices to realize puncture and aspiration of the egg cells.

The duct length difference H between the main input duct position corresponding to the light source device 203 and the egg cell image acquisition device 202 and the main input duct 210 position corresponding to the first valve port 205a is a preset value.

In this way, the central controller 202 calculates, based on the oocyte image information of the oocyte image obtaining apparatus 202, and by combining the flow rate V of the follicular fluid 20 with the oocytes 21 in the main input conduit 210 and the conduit length difference H, the opening/closing time for controlling the first port 205a, the second port 205b and the third port 205c of the sorting switch 205, so that the oocytes 21 in the follicular fluid 20 with the oocytes 21 flow out from the first port 205a, while the follicular fluid 22 without the oocytes 21 flows out from the second port 205b and flows into the follicular fluid collection container 204, and the oocytes 21 flowing out from the first port 205a, namely, the oocytes 24 are combined with the specific culture fluid 23 flowing in from the third port 205c and then flows out to the culture carrier 1.

It can be seen that the automatic egg cell identification and sorting device 200 disclosed in this embodiment can be applied to automatic identification and sorting of cumulus cell complexes, egg cells and early fertilized eggs, thereby improving the accuracy of cell identification and sorting and reducing the operation cost. In addition, the cells sorted by the automatic egg cell identification and sorting device disclosed by the invention are more suitable for in-vitro fertilization, culture and the like.

Referring to fig. 5 to 7, the structure of an egg cell culture carrier 1 is schematically shown in the embodiment. The egg cell culture carrier 1 comprises a carrier body 11, and the carrier body 11 is of a block structure, preferably a square or a rectangle. The surface 111 of the carrier body 11 is provided with a plurality of concave pits 12 which are concave downwards, and the concave pits 12 are used for bearing egg cell liquid drops. Wherein the surface 111 of the carrier body 11 and the surface 121 of each of the pits 12 are both superhydrophobic surfaces. For example, the surface 111 of the carrier body 11 and the surface 121 of each of the pits 12 are both superhydrophobic surfaces on which a layer of superhydrophobic material is laid, or the surface 111 of the carrier body 11 and the surface 121 of each of the pits 12 are both superhydrophobic surfaces subjected to superhydrophobic treatment.

A closed ring-shaped restriction portion 13 is convexly provided on the surface 111 of the carrier body 11 at the edge of each of the recesses 12, and the surface of the restriction portion 13 is an oleophilic surface. The closed annular convex limiting part 13 with oleophilic surface characteristic can limit the overflow of the culture oil outside the egg cell liquid drop, and ensure that the egg cell liquid drop is always in an independent culture environment.

For example, as shown in fig. 7, when fertilization and culture are performed on a culture carrier in which the pit surfaces 121 are all superhydrophobic surfaces, a cell droplet (aqueous solution) 502 containing a cell 501 is spherical on the superhydrophobic surface of the pit surface 121, has a small contact area with the pit surface 121, does not adhere to the surface (corresponding to a suspended state), and can be automatically and precisely positioned on the pit surface 121 (and is the lowest point of the pit surface 121) in a gravitational environment. And a closed ring-shaped limiting part 13 is convexly arranged on the edge of each pit 12, the surface of the limiting part 13 is an oleophilic surface, and the overflow of the culture oil 503 (used for covering the pit surface 121 and the cell droplets 502 arranged in the pit surface 121) outside the cell droplets (aqueous solution) 502 can be limited, so that the cell droplets (aqueous solution) 502 are always kept in an independent culture environment.

In this embodiment, as shown in fig. 6 to 7, the surface 121 of each of the pits 12 is a cambered surface, preferably a semi-spherical surface. The size of each pit 12 is consistent, and the depth and the width of each pit 12 are set to be different according to experimental requirements.

In the present embodiment, the plurality of wells 12 on the carrier body 11 are arranged in a single row on the surface 111 of the carrier body 11, so that the carrier 1 is constituted as a single-row multi-unit culture carrier.

It can be seen that the egg cell culture carrier used in this example has a plurality of pits 12 for holding egg cell droplets on the surface of the carrier 1, and the carrier surface 111 and the pit surface 121 are both superhydrophobic surfaces that have been treated with superhydrophobicity, so that when fertilization and culture are performed on such a culture carrier, the cell droplets (aqueous solution) are spherical on the superhydrophobic surface of the culture carrier, and the contact area with the surface is small, and do not adhere to the surface. In addition, because the concave pit edge on the carrier surface is convexly provided with the closed annular limiting part, and the limiting part has the oleophylic surface characteristic, the overflow of the culture oil outside the cell liquid drops can be limited, and the cell liquid drops are ensured to be always in an independent culture environment. Therefore, the egg cell liquid drop (aqueous solution) can be automatically and accurately positioned to the required position (fertilization and culture) under the gravity environment, thereby facilitating the fertilization and culture operation.

Referring to fig. 8, a schematic diagram of a direct insemination mode device 300 is provided according to an embodiment of the present invention. In this embodiment, the direct insemination mode apparatus 300 includes a first droplet changing operation apparatus including a waste liquid extraction controller 301, a culture liquid replenishment controller 302, a droplet output line 303, a droplet input line 304, and a central controller 9, and both ends of the droplet output line 303 and the droplet input line 304 are connected to a droplet operation needle 305 to suck/output droplets. The waste liquid extraction controller 301 communicates with the droplet discharge line 303, and controls opening/closing of the droplet discharge line 303 and the size of the opening to realize whether to extract the waste liquid in the cell droplets 24 on the culture carrier 1 and to control the flow rate of the extracted waste liquid. The culture solution supplement controller 302 is in communication with the droplet input pipeline 304, and realizes whether to deliver the culture solution (sperm suspension) to the cell droplets on the culture carrier and controls the flow rate of the delivered culture solution (sperm suspension) by opening/closing the droplet input pipeline 304 and controlling the opening size.

The central controller 9 is connected to the waste liquid extraction controller 301 and the culture solution supplement controller 302, respectively, to control the waste liquid extraction controller 301 and the culture solution supplement controller 302 to operate. Specifically, the central controller 9 controls the opening/closing of the liquid drop output pipeline 303 and the opening size to control whether to draw the waste liquid in the egg cell liquid drop on the culture carrier 1 and control the flow rate of the drawn waste liquid by controlling the waste liquid drawing controller 301, and controls the opening/closing of the liquid drop input pipeline 304 and the opening size to control whether to convey the culture liquid (sperm suspension) to the egg cell liquid drop on the culture carrier and control the flow rate of the conveyed culture liquid (sperm suspension) by controlling the culture liquid supplement controller 302.

In this embodiment, the waste liquid extraction controller 301 and the culture liquid supplement controller 302 are peristaltic pumps or stepping motors.

In this embodiment, the direct insemination mode apparatus 300 further comprises a cell image acquisition device 308 connected to said central controller 9. The cell image acquiring device 308 is configured to acquire cell image information in the cell droplets 24 on the culture carrier 1 and send the acquired cell image information to the central controller 9. The central controller 9 processes the cell image information sent by the cell image acquiring device 308 (for example, sequentially stores the image information of each marked position acquired by the cell image acquiring device 308 as an image set of a specific position) to obtain clearer cell image information, and displays the processed cell image information through the display 10. The cell image information of the operator is used for judging whether the cell liquid drops 24 need to be subjected to liquid changing operation, so that the central controller 9 controls the waste liquid extraction controller 301 and the culture liquid supplement controller 302 to perform corresponding work.

In this embodiment, the cell image acquiring device 308 includes an optical fiber cold light source device and a CCD, which are matched with each other, and the optical fiber cold light source device is disposed on the culture carrier 1 to irradiate the cell droplets 24 on the culture carrier 1, so as to form an image on the CCD disposed on the other side of the culture carrier 1 and facing the optical fiber cold light source. The cell image imaged by the CCD is sent to the central controller 9 for image processing to obtain a more complete image.

Preferably, in the first liquid droplet changing operation device of this embodiment, the liquid droplet operation needles 305 connected to both ends of the liquid droplet output pipeline 303 and the liquid droplet input pipeline 304 are all detachable liquid droplet operation needles, and therefore, the first liquid droplet changing operation device of this embodiment further includes a liquid droplet operation needle changing actuation device 306. The droplet-manipulating needle replacing action device 306 is connected to the central controller 9 to receive a control instruction. The droplet-manipulating-needle replacing operation device 306 is used for automatically replacing the droplet-manipulating needles connected to both ends of the droplet outlet line 301 and the droplet inlet line 302. Specifically, before or after the liquid change operation is required to be performed on the cell droplets 24 on the culture carrier 1, the central controller 9 sends a control command to the droplet-manipulating-needle changing operation device 306 to instruct the droplet-manipulating-needle changing operation device 306 to change the droplet-manipulating needles 305 for the corresponding droplet output lines 303 and/or droplet input lines 304.

Preferably, the first droplet replacement operation device of the present embodiment further includes a culture solution selection action device 307 connected to the central controller 9. The culture solution selection action device 307 is used for receiving the instruction of the central controller 9 to move the liquid drop input pipeline 304 to the container 310 containing different culture solutions to obtain the corresponding culture solution (sperm suspension). This embodiment further comprises a waste liquid collection container 311 for collecting waste liquid in the egg cell droplets 24 on the culture carrier 1 drawn out via the droplet output line 303.

It is understood that in the direct insemination mode apparatus 300 of the present embodiment, the cell image acquisition device 308 may be integrated with the first liquid droplet changing operation device.

The direct insemination mode device 300 of the embodiment adopts the first liquid drop liquid change operation device to realize the egg cell fertilization mode, and has the following effects: 1. the automatic continuous replacement of the cell culture solution can be realized in the process of egg cell fertilization culture, and the flow rate of the cell culture solution (sperm suspension) can be adjusted according to the experiment requirements; the liquid can be automatically and intermittently changed; 2. the liquid changing process is completely automated, manual operation is not involved, and the liquid changing control precision is high; 3. the liquid drop can be completely transferred without residue, can be directly connected with a device for manufacturing artificial in-vitro multi-cell type complex living tissue by additive manufacturing and is used as a precursor process device for in-vitro incubation of the artificial living tissue; 4. can be applied to artificial assisted reproduction behaviors and cell culture in a gravity-free environment (such as an extraterrestrial space).

Referring to fig. 9, a block diagram of a vitrified freeze/resuscitation mode apparatus 400 according to an embodiment of the present invention is shown. The freeze/resuscitance mode apparatus 400 comprises a central controller 9, a carrier mechanical actuation means 4, a second droplet exchange means 3, a freeze/resuscitance carrier 6, an operator station 7 carrying said freeze/resuscitance carrier, and a container of freezing medium 8. Wherein:

the freezing/thawing carrier 6 is used for carrying freezing target cells (fertilized egg cell liquid drops) to perform freezing operation, the inner surface of the carrier body is of a sheet structure with an arc surface, a super-hydrophobic surface functional region and a hydrophilic surface functional region are arranged on the inner surface of the carrier body, the periphery of the hydrophilic surface functional region is surrounded by the super-hydrophobic surface functional region, and a liquid drop positioning mark is arranged on the hydrophilic surface functional region. The freezing/thawing carrier 6 with the structure is more beneficial to the transfer and positioning of the egg cell liquid drops, thereby being beneficial to the freezing/thawing operation of the egg cell liquid drops. The specific structure of the carrier will be described in detail later with reference to fig. 10. Wherein,

in this embodiment, the carrier mechanical action means 4 and the second droplet-changing liquid operation means 3 are each connected 9 to the central controller for receiving corresponding freeze/resuscitation control commands.

The carrier robot 4 includes a robot body 401, and a carrier gripping robot 41 and a droplet pickup device 42 (fig. 12) provided on the robot body 401.

The droplet pickup device 42 is used for correspondingly adsorbing and transferring the target cell droplets to/from a specific position of the freezing/resuscitation carrier 6 according to the freezing/resuscitation control instruction.

The carrier clamping action device 41 is used for moving the target cell drops after the liquid drop changing operation on the freezing/thawing carrier together with the freezing/thawing carrier 6 into the freezing medium container 8 according to the freezing/thawing control instruction so as to complete freezing/moving the target cell drops after completing freezing in the freezing medium container 8 together with the freezing/thawing carrier 6 out of the freezing medium container 8 onto the operation table 7.

The second droplet liquid changing operation device 3 is used for performing droplet liquid changing operation on target cell droplets on the freezing/thawing carrier 6 carried on the operation table 7 according to the freezing/thawing control instruction.

As shown in fig. 10, the living cell vitrification freezing/thawing carrier 6 of the present embodiment has a sheet-like structure with a curved inner surface 60 of the freezing/thawing carrier 6. The inner surface of the body 60 of the freezing/thawing carrier 6 is provided with a super-hydrophobic surface functional area 61 and a hydrophilic surface functional area 62, the periphery of the hydrophilic surface functional area 62 is surrounded by the super-hydrophobic surface functional area 61, and the hydrophilic surface functional area 62 is provided with a droplet positioning mark 600 (to help positioning of cell droplets).

In this embodiment, the hydrophilic surface functional region 62 is a circular region. Whereas the superhydrophobic surface functional region 61 is an annular ring-shaped region surrounding the hydrophilic surface functional region 62. Furthermore, the center point of the hydrophilic surface functional region 62 coincides with the lowest point of the carrier body 60. Thereby facilitating the positioning of the cell drops.

The superhydrophobic surface functional region 61 is a superhydrophobic surface layer formed by laying a layer of superhydrophobic material on the inner surface of the carrier or a superhydrophobic surface layer obtained by subjecting partial region of the inner surface of the carrier to superhydrophobic treatment.

Similarly, the hydrophilic surface functional region 62 is a hydrophilic surface layer formed by laying a layer of hydrophilic material on the inner surface of the carrier or a hydrophilic surface layer obtained by subjecting a partial region of the inner surface of the carrier to hydrophilic treatment.

When the vitrified freezing/thawing carrier 6 of the embodiment is used as a carrier for carrying cell droplets to perform cell vitrified freezing/thawing operation, the combination mode of the hydrophilic surface functional area 62 and the super-hydrophobic surface functional area 61 can enable droplets with specified volume to be automatically adsorbed and fixed on the hydrophilic surface functional area 62, and the carrier with a regular arc structure can enable cells contained in the droplets to be automatically positioned on the intersection line of the arc surface and the stage under the gravity environment. The body surface of the freezing/thawing carrier 6 is a cambered surface, and the lowest point of the cambered surface is superposed with the central point of the hydrophilic surface functional region 62 for positioning the cell liquid drop, so that the automatic positioning of the cell liquid drop is facilitated. In addition, since the area surrounding the hydrophilic surface functional region 62 is the superhydrophobic surface functional region 61, the cell liquid drop will not adhere to the surface of the superhydrophobic surface functional region 61, thereby further promoting the cell liquid drop to be positioned on the hydrophilic surface functional region 62 provided with the liquid drop positioning mark.

Fig. 11 shows a schematic view of the structure of the operation table 7 of a vitrification freeze/resuscitation mode device 400 in an embodiment of the present invention. The console 7 is a platform with a plane surface, and a transparent operating area 71 is also arranged on the surface. The transparent operating area 71 is intended to carry the above-mentioned freezing/resuscitation carrier 6. The console 7 of the present embodiment is preferably a movable console 7, which realizes movement of the entire console 7 by providing a connecting moving member. And the moving part connected with the operation table 7 is connected with the central controller 9, and the central controller 9 controls the operation table 7 to move, so that the operation table 7 can be controlled to move to a specific position according to the instruction of the central controller 9 to perform the liquid changing operation of cell freezing/resuscitation.

Fig. 12 is a schematic diagram of the structure of the carrier mechanical actuation means 4 of a freeze/resuscitation mode apparatus 400 according to an embodiment of the present invention. The carrier robot 4 specifically includes a robot body 401, and a carrier gripping and moving device 41 and a droplet pickup device 42 provided on the robot body 401.

The droplet pickup apparatus 42 includes a detachable droplet pickup pipe 421 and a detachable negative pressure suction device 422 communicating with the detachable droplet pickup pipe 421 and controlling adsorption thereof. The droplet pickup unit 42 is fixed to the machine body 401 by the detachable negative pressure suction apparatus 422. The detachable negative pressure suction device 422 correspondingly controls the detachable droplet pickup tube 421 to absorb and transfer target cell droplets to/from a specific position of the freezing/resuscitation carrier 6 (e.g., to the culture carrier 1 or other container) according to the received freezing/resuscitation control command.

The carrier holding action means 41 includes a carrier holding member 411 for holding the freezing/thawing carrier and a fixing bracket 412 for fixing the carrier holding member 411 to the machine body 401. The carrier holding part 411 holds and moves the target cell droplets after the droplet liquid changing operation on the freezing/thawing carrier 6 together with the freezing/thawing carrier 6 into the freezing medium container 8 according to the freezing/thawing control instruction so as to complete freezing/clamp the target cell droplets after completing freezing in the freezing medium container 8 together with the freezing/thawing carrier 6 out of the freezing medium container 8 and move onto the operation table 7.

Preferably, the carrier mechanical action device 4 of this embodiment is a movable carrier mechanical action device 4, the carrier mechanical action device 4 further includes a moving part 43 connected to the machine body, and the carrier mechanical action device 4 is movable on the moving guide 404 through the moving part 43. The moving component 43 connected with the carrier mechanical action device 4 is connected with the central controller 9, and the movement of the carrier mechanical action device 4 is realized under the control of the central controller 9, so that the carrier mechanical action device 4 can be controlled to move to a specific position according to the instruction of the central controller 9 to transfer the cell liquid drops or the freezing/thawing carrier 6 and the like.

Fig. 13 is a schematic diagram of the second operating means 3 for the drip-changing liquid of the freeze/resuscitation mode apparatus according to an embodiment of the present invention. The second droplet changing operation apparatus 3 has a structure substantially identical to that of the first droplet changing apparatus shown in fig. 8, and includes a waste liquid extraction controller 301, a culture liquid replenishment controller 302, a droplet output line 303, and a droplet input line 304, and both ends of the droplet output line 303 and the droplet input line 304 are connected to droplet operation needles 305 for sucking/outputting droplets. The waste liquid extraction controller 301 is communicated with the liquid droplet output pipeline 303, and realizes whether to extract waste liquid in the cell liquid droplets on the freezing/thawing carrier 6 and controls the flow rate of the extracted waste liquid by controlling the opening/closing of the liquid droplet output pipeline 303 and the size of the opening. And the culture solution supplement controller 302 is communicated with the liquid drop input pipeline 304, and realizes whether to convey the culture solution into the egg cell liquid drops on the freezing/thawing carrier 6 and controls the flow rate of the conveyed culture solution through the opening/closing of the liquid drop input pipeline 304 and the control of the opening size.

The central controller 9 is connected to the waste liquid extraction controller 301 and the culture solution supplement controller 302, respectively, to control the waste liquid extraction controller 301 and the culture solution supplement controller 302 to operate. Specifically, the central controller 9 controls the opening/closing of the droplet output line 303 and the opening size to control whether to draw the waste liquid from the cell droplets on the freezing/thawing carrier 6 and control the flow rate of the drawn waste liquid by controlling the waste liquid drawing controller 301, and controls the opening/closing of the droplet input line 304 and the opening size to control whether to transfer the culture liquid to the cell droplets on the culture carrier and control the flow rate of the transferred culture liquid by controlling the culture liquid replenishment controller 302. In this embodiment, the waste liquid extraction controller 301 and the culture liquid supplement controller 302 are peristaltic pumps or stepping motors.

Preferably, in the second liquid droplet changing operation device 3 of the present embodiment, the liquid droplet operation needles 305 connected to both ends of the liquid droplet output pipeline 303 and the liquid droplet input pipeline 304 are detachable liquid droplet operation needles, and therefore, the second liquid droplet changing operation device of the present embodiment further includes a liquid droplet operation needle changing actuation device 306. The droplet-manipulating needle replacing action device 306 is connected to the central controller 9 to receive a control instruction. The droplet-manipulating-needle replacing operation device 306 is used for automatically replacing the droplet-manipulating needles connected to both ends of the droplet outlet line 301 and the droplet inlet line 302. Specifically, before or after the liquid change operation is required to be performed on the cell droplets on the freezing/resuscitation carrier 6, the central controller 9 sends a control instruction to the droplet-manipulating-needle replacement actuating device 306 to instruct the droplet-manipulating-needle replacement actuating device 306 to replace the droplet-manipulating needle 305 with the corresponding droplet output pipeline 303 and/or droplet input pipeline 304.

Preferably, the second droplet-changing operation device 3 of the present embodiment further includes a culture medium selection operation device 307 connected to the central controller 9. The culture fluid selection action device 307 is used for receiving the instruction of the central controller 9 to move the liquid drop input line 304 to the container 310 containing different culture fluids to obtain the corresponding freezing/thawing fluid (i.e. cryoprotectant/thawing fluid). The second droplet replacement operation apparatus of the present embodiment further includes a waste liquid collection container 311 for collecting waste liquid in the egg cell droplets on the freezing/thawing carrier 6 drawn out via the droplet output line 303.

Referring back to fig. 1, the freezing medium contained in the freezing medium container 8 of the present embodiment is liquid nitrogen.

In addition, the automatic operation device for vitrification freezing/resuscitating living cells of the present embodiment further includes an image acquisition device 308b and a display 10 connected to the central controller 9, respectively. The image acquiring device 308b is configured to acquire image information of egg cells in the cell droplets on the freezing/resuscitation carrier 6, send the acquired image information of the egg cells to the central controller 9, process the processed image information, and display the processed image information on the display 10. Therefore, the droplet positioning marks on the hydrophilic surface functional region 62 of the freezing/thawing carrier 6 can be recognized by the image information acquired by the image acquiring means 308b, thereby positioning the positions where the egg cell droplets are placed.

In this embodiment, the image acquiring device 308b is formed by the optical fiber cold light source device and the cell image acquiring device CCD. The optical fiber cold light source device is arranged right above the operation table 7 and is opposite to the egg cell liquid drops on the freezing/recovering carrier 6 on the transparent operation area 71 of the operation table 7, and the cell image acquisition device CCD is arranged right below the operation table 7 and is opposite to the optical fiber cold light source device. The light source device 20 illuminates the cell droplets on the freezing/thawing carrier 6 on the transparent operating area 71 of the operation table 7 and images them on the cell image acquiring device CCD. It is understood that the image capturing device 308b of the present embodiment may also be integrated into the second droplet-changing liquid operation device 3, and is integrated with the second droplet-changing liquid operation device 3.

It can be seen that, in the cellular vitrification freezing/resuscitation mode device of this embodiment, the carrier of the sheet structure with the inner surface in the arc surface is used as the freezing/resuscitation carrier, the inner surface of the freezing/resuscitation carrier is provided with the superhydrophobic surface functional region and the hydrophilic surface functional region, the periphery of the hydrophilic surface functional region is surrounded by the superhydrophobic surface functional region, and the hydrophilic surface functional region is provided with the droplet positioning identifier, so that in the whole freezing/resuscitation automatic operation process, the whole freezing/resuscitation operation process of the freezing target cell can be automatically completed through the identification and positioning of the droplet positioning identifier, the manual participation is not needed, the work efficiency is greatly improved, and the stability, timeliness and safety of the operation are ensured.

It will be appreciated that the transplant mode device located within the sub-culture housing 102 in the automated device for in vitro fertilization and cleavage culture of oocytes may perform the transplanting function by the vitrification freezing/resuscitation mode device 400.

In the following, how to implement the working processes of the in vitro fertilization and cleavage culture of the egg cells including, but not limited to, a cumulus cell identification and sorting mode, a direct insemination mode, a freezing mode, a freeze-thaw cell resuscitation mode, a transplantation mode and the like by using the automatic device for the in vitro fertilization and cleavage culture of the egg cells provided by the embodiment of the invention is described in detail.

Cumulus cell identification and sorting mode

Referring to fig. 1 to 3, the operator inputs the expected number of eggs to enter the cumulus complex sorting mode by outputting an instruction to the egg cell automatic identification sorting apparatus 200 through the central controller 9, and the micro-fluid pump pumps the culture solution with a volume corresponding to the expected number of eggs. And the central controller 9 instructs the culture carrier drive device 105 to operate to bring the carried culture carrier 1 into a prescribed initial position.

First, the central controller 209 controls the second valve port 205b of the sorting switch 205 to open (when the first valve port 205a and the third valve port 205c are closed), and controls the negative pressure generator 201 to start. Upon activation of the negative pressure generator 201, the follicular fluid 20 with the ova 21 flows into the main inlet conduit 210 under the negative pressure.

When the follicle stock solution 20 with the ova 21 flows through the main input conduit 210 and flows through the oocyte image acquisition device 202, the light source device 203 cooperates with the oocyte image acquisition device 202 to acquire oocyte image information.

The oocyte image obtaining device 202 sends the obtained oocyte image information to the central controller 209, the central controller 209 calculates the precise time for the follicle stock solution 20 with the oocytes 21 to reach the first valve port 205a according to the oocyte image information and by combining the flow rate V of the follicle stock solution 20 with the oocytes 21 in the main input conduit 210 and the conduit length difference H, and when the follicle stock solution 20 with the oocytes 21 reaches the first valve port 205a, the central controller 209 immediately controls the sorting switch 205 to open the first valve port 205a (at this time, the second valve port 205b and the third valve port 205c are closed), so that the oocytes 21 in the follicle stock solution 20 with the oocytes 21 flow out from the first valve port 205a and enter the main output conduit 211. And immediately after the egg 21 flows out of the first valve port 205a and enters the main outlet pipe 211, the central controller 209 controls the sorting switch 205 to open the third valve port 205c (at this time, the first valve port 205a and the second valve port 205b are closed), so that the specific culture medium 23 flowing in from the auxiliary inlet pipe 213 and the egg 21 form an egg drop 24, and then flows out through the main outlet pipe 211 and flows into the culture medium 1 in the automatic interconnected partition combination culture box 100 through the channel 106. When the specific culture medium 23 flowing in from the secondary input conduit 213 reaches a predetermined volume, the central controller 209 immediately controls the sorting switch 205 to open the second valve port 205b (at this time, the first valve port 205a and the third valve port 205c are closed), and causes the follicular fluid 22 from which the ova 21 have been removed to flow out from the second valve port 205b into the follicular fluid collection container 204.

The central controller 9 controls the device to repeat the action series of image recognition, valve opening and closing of the sorting switch 205, pumping of liquid drops by the micro-flow pump 207 and sequential entering of ovum fine liquid drops into a specific culture position (culture carrier 1) until the negative pressure generator 201 is closed and the sorting mode of the ovum dune cell compound is ended.

In addition, after termination of the cumulus cell complex sorting mode, the central controller 9 controls to open the automatic airtight door 103 and instructs the culture carrier driving means 105 to operate to transfer the carried culture carrier 1 from the sub-culture casing 102 into the main culture casing 101 (and the central controller 9 controls to close the automatic airtight door 10) to wait for the direct fertilization mode operation.

It is understood that the central controller 9 may control the first/second droplet changing operation device to change the liquid of the egg cell droplet on the culture carrier 1 to cover the egg cell droplet with a specific amount of culture oil before the direct fertilization mode operation is performed.

Direct fertilization model

The central controller 9 controls an environment management device built in the main culture box 101 to adjust the environment in the main culture box 101 so as to meet the environmental requirements of the egg cell droplet fertilization mode, when the egg cell droplets on the culture carrier 1 enter a specific position in the main culture box 101 for fertilization and culture operation, the central controller 9 controls the direct insemination mode device 300 (first droplet liquid changing operation device) to immediately and sequentially and automatically change the liquid for the egg cell droplets on the culture carrier 1, and records the image information of the oocytes entering the in vitro culture state at a set frequency through the cell image acquisition device 308.

Clear image information acquired by the cell image acquisition equipment 308 is sent to the central controller 9, and the central controller 9 successively stores the image information of each marked position as an image set of a specific position; the operator can read the image set at any marked position at any time, judge the development state of the cumulus cell complex and record the development state;

the operator determines the time for adding the sperm suspension according to the egg cell state displayed by the image set and instructs the central controller; the central controller 9 controls the first drop changing operation device to add sperm suspension with a specified volume to the oocyte drops on the culture carrier 1 according to the instruction, thereby completing insemination.

Cell vitrification freezing mode

After the egg drop has completed the fertilization mode, the operator can open the automatic airtight door 103 under the control of the central controller 9 and instruct the culture carrier driving device 105 to operate to transfer the carried culture carrier 1 from the sub-culture chamber 102 into the sub-culture chamber 102 (and the central controller 9 controls the automatic airtight door 10 to be closed) to wait for other modes of operation.

When the operator sends a plurality of control commands to enter the cell freezing mode through the central controller 9, the environment management device built in the sub-incubator 102 first receives the control commands sent by the central controller 9, and correspondingly adjusts the environment in the sub-incubator 102 to the state of the egg cell freezing operation. Then, the moving means 43 of the carrier robot 4 moves the carrier robot 4 to an appropriate position (so that the detachable droplet pickup tube 421 of the droplet pickup device 42 is directed to the target cell droplet on the culture carrier 1, that is, the disrupted cell droplet after fertilization) in accordance with the received control command. Then, the droplet pickup device 42 controls the detachable droplet pickup tube 421 to adsorb the target cell droplets from the culture carrier 1 and transfer the target cell droplets onto a specific position (i.e., the hydrophilic surface functional region 62 provided with the droplet positioning marks) of the freezing/thawing carrier 6 on the operation table 7 (specifically, the specific position is positioned by the image acquisition device 308 b) according to the received control instruction. Then, the moving component of the console 7 moves the console 7 carrying the freezing/thawing carrier 6 (and the cell droplets) to the operation zone of the droplet liquid changing device according to the received control command (specifically, the image obtaining device 308b locates the droplet positioning mark on the freezing/thawing carrier 6, and the console 7 is moved to make the cell droplets carried by the console 7 fall on the operation point of the droplet liquid changing device), and then, the second droplet liquid changing device 3 performs the liquid changing operation of cell freezing on the cell droplets on the specific position of the freezing/thawing carrier 6 on the console 7 according to the received control command, and changes the droplet components (for example, the cryoprotectant) according to the predetermined program. At this time, the recording cell image information is acquired by the image acquisition device 308 b. Until the target cells on the freezing/thawing carrier 6 reach the equilibrium state with the cryoprotectant and the droplet volume reaches the specified value range (i.e. when the surface area of the cells exposed to the environment reaches the preset value), the moving part 43 of the carrier mechanical action device 4 moves the carrier mechanical action device 4 to the proper position (the carrier holding part 411 of the carrier holding action device 41 is opposite to the target cell droplet) according to the received control instruction, and the carrier holding action device 41 holds and rapidly moves the target cell droplet on the freezing/thawing carrier 6 after the droplet liquid changing operation is completed into the freezing medium container 8 (for example, liquid nitrogen) together with the freezing/thawing carrier 6 to complete the freezing.

Cell vitrification Resuscitation mode

When a plurality of control commands for entering the cell recovery mode are issued by the central controller 9, the environment management device built in the sub-incubator 102 first receives the control commands issued by the central controller 9, and adjusts the environment in the sub-incubator 102 to a state of the composite cell recovery operation correspondingly. Then, the moving means 43 of the carrier robot 4 moves the carrier robot 4 to a proper position (so that the carrier holding means 411 of the carrier holding robot 41 faces the target cell droplet) according to the received control command, and then the carrier holding robot 41 controls the carrier holding means 411 to pick up the target cell droplet frozen in the freezing medium container 8 together with the freezing/thawing carrier 6 from the freezing medium container 8 and move the target cell droplet onto the operation table 7 according to the received control command. Then, the moving component of the console 7 moves the console 7 carrying the freezing/thawing carrier 6 (and the cell droplets) to the operation zone of the droplet liquid changing device according to the received control command (specifically, the image acquisition device 308b positions the droplet positioning marks on the freezing/thawing carrier 6, and the console 7 is moved to make the cell droplets carried by the console 7 fall on the operation point of the droplet liquid changing device), and then, the second droplet liquid changing device 3 performs the liquid changing operation of cell thawing on the cell droplets at the specific position of the freezing/thawing carrier 6 on the console 7 according to the received control command, and changes the liquid volume and the liquid components according to the predetermined program. At this time, the recording cell image information is acquired by the image acquisition device 308 b. Until the cells reach the specified equilibrium state of the culture solution, the recovery is completed, the moving part 43 of the carrier mechanical action device 4 moves the carrier mechanical action device 4 to a proper position (the detachable droplet pickup tube 421 of the droplet pickup device 42 is aligned with the target cell droplet) according to the received control command, and the detachable droplet pickup tube 421 adsorbs and transfers the target cell droplet on the freezing/recovering carrier 6 after the droplet changing operation is completed (for example, transfers the target cell droplet to the culture carrier 1 or another container) according to the received control command.

Migration mode

After the egg drop has completed the fertilization mode, if the operator decides to perform the transplantation, he can first open the automatic airtight door 103 through the central controller 9 and instruct the culture carrier driving device 105 to operate to transfer the loaded culture carrier 1 from the sub-incubator 102 to the sub-incubator 102 (and the central controller 9 controls to close the automatic airtight door 10).

The operator can enter the transplanting mode by the instruction of the central controller 9, and first, the second droplet replacement operation means 3 performs a fluid replacement operation on the egg cell droplets of the culture carrier 1 in accordance with the received control instruction, and changes the liquid volume and composition in accordance with a predetermined program. At this time, the recording cell image information is acquired by the image acquisition device 308 b. When the liquid drop on the cell reaches the predetermined volume, the moving part 43 of the carrier mechanical action device 4 moves the carrier mechanical action device 4 to a proper position (the detachable liquid drop pickup tube 421 of the liquid drop pickup device 42 is aligned with the target cell liquid drop) according to the received control command, and the detachable liquid drop pickup tube 421 sucks the egg cell liquid drop on the freezing/thawing carrier 6 after the liquid drop changing operation is completed and quickly loads the egg cell liquid drop into the transplantation tube according to the received control command, thereby completing the transplantation mode.

Another embodiment of the present invention provides an automated method for in vitro fertilization and cleavage culture of egg cells, wherein a plurality of operation modes including a cumulus complex identification and sorting mode, a direct insemination mode, a vitrification freezing/thawing mode and a transfer mode are all used for operating egg cell droplets, including but not limited to identification and sorting of egg cell droplets, liquid changing of egg cell droplets and transfer and transplantation of egg cell droplets.

While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.

Claims

1. An automatic device for in-vitro fertilization and cleavage culture of egg cells is characterized by comprising an automatic communication type partitioned combined incubator, a central controller and an automatic egg cell identification and sorting device, wherein the central controller is arranged outside the incubator;

the automatic communication type subarea combined incubator is provided with operation mode devices at different stages in the process of fertilization of egg cell liquid drops and culture of cleavage in different subareas, and each subarea is communicated/separated through an automatic airtight door; the automatic communication type partitioned combined incubator is internally provided with a culture carrier for bearing egg cell liquid drops and a culture carrier driving device for driving the culture carrier to move to and fro in each partition, the surface of the culture carrier is provided with a plurality of pits for bearing the egg cell liquid drops, and the surface of each pit is a super-hydrophobic surface;

2. The automated apparatus for in vitro fertilization and cleavage culture of egg cells according to claim 1, wherein the automated, intercommunicated, partitioned incubator comprises two partitions consisting of a main incubator body, a sub-incubator body, and the automated airtight door for communication/separation between the main and sub-incubator bodies.

3. The automated apparatus for egg cell in vitro fertilization and cleavage culture according to claim 1, wherein the operation mode apparatus comprises a direct insemination mode apparatus, a vitrification freezing/resuscitation mode apparatus, and a transplantation mode apparatus.

4. The automated apparatus for in vitro fertilization and cleavage culture of egg cells according to claim 3, wherein the direct insemination mode apparatus comprises a first droplet exchange operation apparatus, the first droplet exchange operation apparatus comprises a waste liquid extraction controller, a culture liquid replenishment controller, a droplet output pipeline and a droplet input pipeline, and both ends of the droplet output pipeline and the droplet input pipeline are connected to droplet operation needles for sucking/outputting droplets; the waste liquid extraction controller extracts waste liquid in cell drops on the culture carrier through the drop output pipeline and controls the flow of the extracted waste liquid, and the culture liquid supplement controller conveys culture liquid to the cell drops on the culture carrier through the drop input pipeline and controls the flow of the conveyed culture liquid; and the central controller is respectively connected with the waste liquid extraction controller and the culture solution supplement controller so as to control the waste liquid extraction controller and the culture solution supplement controller to work.

5. The automated apparatus for egg cell in vitro fertilization and cleavage culture according to claim 4, wherein the waste fluid extraction controller and the culture fluid replenishment controller are peristaltic pumps or stepper motors.

6. The automated apparatus for egg cell in vitro fertilization and cleavage culture according to claim 4, wherein the direct insemination mode apparatus further comprises a cell image acquisition device coupled to the central controller for acquiring cell image information in cell droplets on the culture carrier during direct insemination mode operation.

7. The automated apparatus for egg cell in vitro fertilization and cleavage culture according to claim 1, wherein a closed ring-shaped restriction is convexly provided on the surface of the culture carrier at the edge of each of the pits, and the surface of the restriction is an oleophilic surface.

8. The automated apparatus for in vitro fertilization and cleavage culture of egg cells according to claim 1, wherein the automatic egg cell identification and sorting apparatus comprises a negative pressure generator, an egg cell image acquisition device, a light source device, a follicular fluid collection container and a sorting switch, and the central controller is respectively connected with the negative pressure generator, the egg cell image acquisition device and the sorting switch;

9. The automated apparatus for oocyte in vitro fertilization and cleavage culture according to claim 8, wherein the central controller controls operation of the negative pressure generator to control a flow rate of the follicular fluid with the oocytes in the main input conduit.

10. The automated apparatus for egg cell in vitro fertilization and cleavage culture according to claim 9, wherein the difference between the main input conduit position corresponding to the light source device and the egg cell image obtaining device and the main input conduit position corresponding to the first valve port has a preset conduit length.

11. The automated apparatus for oocyte in-vitro fertilization and cleavage culture according to claim 10, wherein the central controller calculates the on/off time of the first, second and third ports of the sorting switch according to the image information of the oocyte and by combining the flow rate of the follicle stock solution with the oocyte in the main input conduit and the conduit length difference.

12. The automated apparatus for egg cell in vitro fertilization and cleavage culture according to claim 3, wherein the vitrification freezing/resuscitating mode device comprises a carrier mechanical action device, a second droplet exchange device, a freezing/resuscitating carrier, a station carrying the freezing/resuscitating carrier, and a freezing medium container, wherein,

the carrier mechanical action device comprises a mechanical body, and a carrier clamping action device and a liquid drop pickup device which are arranged on the mechanical body; the liquid drop pickup device is used for correspondingly adsorbing and transferring target cell liquid drops to a specific position of the freezing/resuscitation carrier or adsorbing and transferring the target cell liquid drops from the freezing/resuscitation carrier according to a freezing/resuscitation control instruction; the carrier clamping action device is used for moving the target cell drops after the liquid drop changing operation on the freezing/thawing carrier and the freezing/thawing carrier into the freezing medium container together according to a freezing/thawing control instruction so as to complete freezing/moving the target cell drops after freezing in the freezing medium container and the freezing/thawing carrier from the freezing medium container to the operation table;

13. The automated apparatus for egg cell in vitro fertilization and cleavage culture according to claim 12, wherein the vitrification freezing/resuscitation mode apparatus further comprises an image acquisition device connected to the central controller; the image acquisition device is used for acquiring cell image information in the cell liquid drops on the freezing/resuscitation carrier and sending the acquired cell image information to the central controller for processing.

14. The automated apparatus for egg cell in-vitro fertilization and cleavage culture according to claim 12, wherein the carrier body of the freezing/thawing carrier is a sheet structure with an inner surface having a cambered surface, the inner surface is provided with a superhydrophobic surface functional region and a hydrophilic surface functional region, the periphery of the hydrophilic surface functional region is surrounded by the superhydrophobic surface functional region, and the hydrophilic surface functional region is provided with a droplet positioning mark.

15. The automated apparatus for egg cell in vitro fertilization and cleavage culture according to claim 14, wherein a center point of the hydrophilic surface functional region coincides with a lowest point of the carrier body.

16. The automated apparatus for egg cell in vitro fertilization and cleavage culture according to claim 3, wherein the transplantation mode apparatus performs a transplantation function by the vitrification freezing/resuscitation mode apparatus.

17. The automated apparatus for in vitro fertilization and cleavage culture of egg cells according to claim 2, wherein the main/sub-incubator respectively comprises an environmental management device for controlling and adjusting the temperature, humidity, pressure and partial pressure of gas in the main/sub-incubator.

18. The automated apparatus for in vitro fertilization and cleavage culture of egg cells according to claim 2, wherein the main/sub culture housings are provided with a flow path communicating with the outside, and the flow path receives a drop of cells to be cultured from the outside or sends a drop of cells after culture to the outside.