CN104396942A - Automatic operation method, system and carrier of live cell vitrification freezing/thawing - Google Patents
Automatic operation method, system and carrier of live cell vitrification freezing/thawing Download PDFInfo
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- CN104396942A CN104396942A CN201410626064.2A CN201410626064A CN104396942A CN 104396942 A CN104396942 A CN 104396942A CN 201410626064 A CN201410626064 A CN 201410626064A CN 104396942 A CN104396942 A CN 104396942A
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Abstract
The invention discloses a live cell vitrification freezing/thawing carrier. The carrier body is a transparent sheet with an arc-shaped internal surface, which is provided with an ultra-hydrophobic surface functional area and a hydrophilic surface functional area, wherein the periphery of the hydrophilic surface functional area is surrounded by the ultra-hydrophobic surface functional area, and the hydrophilic surface functional area is provided with droplet positioning labels. The invention further discloses an automatic operation device and an automatic operation method of live cell vitrification freezing/thawing.
Description
Technical field
The present invention relates generally to vitrification field, particularly relates to method of automatically operating and the system of the glass freezing/recovery of a kind of active somatic cell, and active somatic cell glass freezing/recovery carrier.
Background technology
In human assistance reproductive technology, the storage in vitro of human reproduction's power is always extensively concerned.Up to now, human sperm bank normal operation is close to 30 years, the warm resuscitation technique of glass frozen of fertilized egg starts to popularize in this century in early days, but the storage in vitro of human oocytes is limited by the limitation of method of operating, there is no substantive progress, in global range, also do not drop into mankind's ovum of stable operation and application.Compare from physiological sources, mankind's ovum is the main object of human reproduction's power storage in vitro, and sperm takes second place; The storage in vitro service of fertilized egg to as if family, the fertilized egg of storage in vitro, ethics and obtained not modifiable attribute is legally the result of reproductive function, or perhaps the frozen state of reproductive behavio(u)r and intermediateness.
The freezing and thawing technology of egg cell is different from the cryopreservation resuscitation of body early embryo and sperm, require stricter to the success rate of freezing and thawing, the physiology formation of egg cell itself is different from embryonic cell, and current method and apparatus is not enough to reach egg cell standardization freeze thawing and stable recovery.
The warm resuscitation technique of now widely used body early embryo glass frozen relies on manual operations.Frozen-thaw process comprises: 1, operator makes the dehydrating agent of glass freezing and the combination of cryoprotector drop, prepares freezing carrier and marks, registers; 2, adjust between operating environment temperature to Suitable Area; 3, manual absorption needs freezing cell, 4, transitional cell to dehydrating agent drop, wait cell dehydration is to suitable state, and generally this process assert whether reach standard with the average dehydration time; 5, fast transfer cell enters cryoprotector drop, maintains the sufficiently long time; 6, fast transfer cell is to freezing carrier, and draw the liquid of cell peripheral absorption fast, the surface area that cell is exposed is maximum; 7, the carrier being stained with cell is dropped into liquid nitrogen fast, tubulature, record, complete operation.
The operation of vitrifying freeze thawing cell recovery relies on gradient resuscitation fluid system, sequence resuscitation fluid is designed to different concentration gradients, comprises step: 1, taken out by the carrier in liquid nitrogen environment, be placed into gradient liquid one fast, identify and confirm cell detachment carrier, rewarming; 2, according to the time series of setting, transitional cell is to gradient liquid two, three, four successively; 3, washing and transitional cell to culture fluid; 4, observe and record cell state, completing resuscitation.
Glass freezing and the recovery of egg cell cannot reach standardization under manual state.Due to structure and the composition characteristics of cell-specific, egg cell can not tolerate dehydration fast, can not tolerate the Thief zone pressure reduction required by fast dewatering, and safety reaches resting state.Manual freezing egg cell, must dewater and cryoprotector liquid system by implementation sequence, and the concentration gradient difference of sequence liquid need meet the physiological requirement of egg cell dehydration.Under pattern hand-manipulated, operator wants be shifted successively in a large amount of sequence drops by cell fast, or gradient liquid is arranged to the drop that closes on mutually, after cell being transferred to homing sequence drop, at official hour, initial droplet is communicated with the second sequence drop, due to permeable pressure head between drop, natural one-way flow merges, by signaling to drop communicating passage, egg cell enters the second drop naturally, successively after operation, be quickly transferred into liquid nitrogen environment, complete freezing.And the resuscitation process of freeze thawing egg cell is the repetition of foregoing freeze process, but the order of gradient liquid is contrary.
Egg cell shifts successively fast by operator in a large amount of sequence drop, strict to the skill set requirements of operator, can not avoid maloperation and operate miss; In drop communicating method, the liquid environment of cell is not controlled, very strict to the skill set requirements of operator yet, and operating result cannot standardization, and this has had a strong impact on the clinical practice of the freezing and recovery of egg cell.If the success rate of egg cell freeze thawing recovery can not close to 95% even closer to 100%, the practicality of this method of operating will be under suspicion, because the limited source of egg cell, after a treatment cycle, the egg cell quantity obtained can not be too many, in some cases, each operation can only obtain 1 available egg cell (microstimulation scheme, free period get ovum scheme etc.); If need freezing egg cell quantity many, the physical efficiency requirement of continuous print repeatable operation process to operator can raise, and affects operating result.
Therefore, method of automatically operating and the system of developing a kind of active somatic cell glass freezing is needed.
Summary of the invention
The object of this invention is to provide method of automatically operating and the system of the glass freezing/recovery of a kind of active somatic cell, and active somatic cell glass freezing/recovery carrier, automatically can complete and include but not limited to that the automatic glassization of the active somatic cells such as egg cell, body early embryo, blastaea is freezing and recover, and provide standardized operating process.
For achieving the above object, The embodiment provides a kind of active somatic cell glass freezing/recovery carrier, the laminated structure of described carrier body to be inner surface be cambered surface, the inner surface of described carrier body is provided with super hydrophobic surface functional area and water-wetted surface functional area, super hydrophobic surface functional area described in described water-wetted surface functional area four periderm around, described water-wetted surface functional area is provided with drop positioning mark.
As the improvement of technique scheme, described super hydrophobic surface functional area is circular annular region, and described water-wetted surface functional area is border circular areas.
As the improvement of technique scheme, the central point of described water-wetted surface functional area overlaps with the minimum point of described carrier body.
As the improvement of technique scheme, described super hydrophobic surface functional area is on the inner surface of described carrier, laid the super hydrophobic surface layer of one deck super hydrophobic material or described super hydrophobic surface functional area is by the super hydrophobic surface layer of the subregion of the inner surface of described carrier gained after super-hydrophobic process.
As the improvement of technique scheme, described water-wetted surface functional area is on the inner surface of described carrier, laid the hydrophilic surface layer that the hydrophilic surface layer of one deck water wetted material or described water-wetted surface functional area are the subregion gained after hydrophilic treated for the inner surface by described carrier.
The embodiment of the present invention additionally provides the automatic pilot of a kind of active somatic cell glass freezing/recovery, comprise central controller, carrier mechanical action device, drop change liquid operating means, freezing/recovery carrier, carry described freezing/operating desk of recovery carrier and refrigerant container, wherein
Described carrier mechanical action device and described drop change liquid operating means be connected with described central controller respectively to receive corresponding freezing/recovery control instruction;
Described carrier mechanical action device comprises basic machine and the carrier holding action device that is arranged on basic machine and drop pick-up unit; Described drop pick-up unit for according to freezing/recovery control instruction corresponding target cell drop adsorbed and transfer to described freezing/ad-hoc location of recovery carrier/by target cell drop from described freezing/recovery carrier adsorbs and shifts away; Described carrier holding action device be used for according to freezing/recovery control instruction, by described freezing/recovery carrier on complete drop change liquid operation after target cell drop together with described freezing/recovery carrier be moved in described refrigerant container with complete freezing/be moved out on described operating desk together with freezing/recovery carrier by completing freezing target cell drop in described refrigerant container from described refrigerant container;
Described drop change liquid operating means for according to freezing/recovery control instruction, described operating desk is carried freezing/recovery carrier on target cell drop carry out drop change liquid operation.
As the improvement of technique scheme, described freezing/recovery carrier is active somatic cell as above glass freezing/recovery carrier; Described ad-hoc location for described in be provided with on the water-wetted surface functional area of drop positioning mark.
As the improvement of technique scheme, also comprise the image acquiring device and display that are connected with described central controller respectively;
Described image acquiring device for obtain described freezing/recovery carrier on cell drop in cell image information, and after the cell image information got is sent to described central controller process, to be shown by described display.
As the improvement of technique scheme, described image acquiring device can change liquid operating means with described drop and be structure as a whole.
As the improvement of technique scheme, described carrier mechanical action device is packaged type carrier mechanical action device, described carrier mechanical action device also comprises the moving-member be connected with described basic machine, and described carrier mechanical action device can be moved by described moving-member on motion guide rail.
As the improvement of technique scheme, the fixed support that described carrier holding action device comprises the carrier hold assembly for clamping freezing/recovery carrier and is fixed on by described carrier hold assembly on described basic machine.
As the improvement of technique scheme, described drop pick-up unit comprises detachable drop and picks up pipe and pick up pipe with described detachable drop and be communicated with and control its detachable negative-pressure ward equipment be connected.
As the improvement of technique scheme, the refrigerant that described refrigerant container content has is liquid nitrogen.
As the improvement of technique scheme, described automatic operation system is placed in the controlled cell culture incubator of environment.
The embodiment of the present invention additionally provides a kind of method of automatically operating of active somatic cell glass freezing, comprises step:
S1, sent the multiple control instructions entering cell freezing pattern to multiple actuating unit by central controller;
S2, by drop pick-up unit and according to the control instruction that receives, on the ad-hoc location of the freezing carrier that target cell drop absorption migration is carried to operating desk;
S3, to be changed liquid operating means by drop and according to the control instruction received, drop is carried out to the target cell drop on the ad-hoc location of the freezing carrier that described operating desk carries and changes liquid operation, until the target cell on described freezing carrier reaches reach specified value scope with the poised state of cryoprotector and drop volume; And
S3, by carrier holding action device and according to the control instruction that receives, by described freezing carrier complete drop change liquid operation after target cell drop be moved in described refrigerant container freezing to complete together with described freezing carrier.
The embodiment of the present invention additionally provides the method for automatically operating of a kind of active somatic cell glass recovery, comprises step:
S1, sent the multiple control instructions entering cell recovery pattern to multiple actuating unit by central controller;
S2, according to the control instruction that receives, be moved out on operating desk together with freezing carrier by completing freezing target cell drop in refrigerant container from described refrigerant container by carrier holding action device; Wherein, described target cell drop is placed on the ad-hoc location of described freezing carrier;
S3, to be changed liquid operating means by drop and according to the control instruction received, drop is carried out to the target cell drop on the ad-hoc location of the freezing carrier that described operating desk carries and changes liquid operation, until the target cell on described freezing carrier reaches the poised state with culture fluid, complete recovery; And
S4, according to the control instruction that receives by drop pick-up unit, described target cell drop is adsorbed from described freezing carrier and shifts away.
Compared with prior art, the method of automatically operating of a kind of active somatic cell glass freezing/recovery that the embodiment of the present invention provides and system, and active somatic cell glass freezing/recovery carrier, by adopting inner surface to be that the carrier of the laminated structure of cambered surface is as freezing/recovery carrier, and the inner surface of this freezing/recovery carrier is provided with super hydrophobic surface functional area and water-wetted surface functional area, super hydrophobic surface functional area described in described water-wetted surface functional area four periderm around, described water-wetted surface functional area is provided with drop positioning mark, like this, when using carrier mechanical action device to carry out the transfer of cell drop, can be adsorbed by cell drop fast and accurately and shift navigates on the ad-hoc location being provided with drop positioning mark of this freezing carrier, thus be beneficial to the cell drop carrying out automation change liquid operation, and whole operating process sends instruction control by central controller, a kind of standardized operating environment and operating process are provided, meet and include but not limited to egg cell, body early embryo, the freezing physiological requirement with recovering of automatic glassization of the active somatic cells such as blastaea, avoid operate miss, standardized freezing and recovery result can be reached, for the operation of mankind's ovum provides infrastructural support.
Accompanying drawing explanation
In order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, be briefly described to the accompanying drawing used required in embodiment or description of the prior art below, apparently, accompanying drawing in the following describes is only some embodiments of the present invention, for those of ordinary skill in the art, under the prerequisite not paying creative work, other accompanying drawing can also be obtained according to these accompanying drawings.
Fig. 1 is the structured flowchart of the automatic pilot of a kind of active somatic cell glass freezing/recovery in the embodiment of the present invention.
Fig. 2 is the structural representation of a kind of active somatic cell glass freezing/recovery carrier in the embodiment of the present invention.
Fig. 3 is the structural representation of the operating desk of the automatic pilot of a kind of active somatic cell glass freezing/recovery in the embodiment of the present invention.
Fig. 4 is the structural representation of the carrier mechanical action device of the automatic pilot of a kind of active somatic cell glass freezing/recovery in the embodiment of the present invention.
Fig. 5 is the structural representation that the drop of the automatic pilot of a kind of active somatic cell glass freezing/recovery in the embodiment of the present invention changes liquid operating means.
Fig. 6 is the flow chart of the method for automatically operating of a kind of active somatic cell glass freezing in the embodiment of the present invention.
Fig. 7 is the flow chart of the method for automatically operating of a kind of active somatic cell vitrifying recovery in the embodiment of the present invention.
Embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, be clearly and completely described the technical scheme in the embodiment of the present invention, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
With reference to figure 1, it is the structured flowchart of the automatic pilot of a kind of active somatic cell glass freezing/recovery in the embodiment of the present invention.This automatic operation system comprises central controller 9, carrier mechanical action device 4, drop change liquid operating means 3, freezing/recovery carrier 6, carry described freezing/operating desk 7 of recovery carrier and refrigerant container 8.Wherein:
Described freezing/recovery carrier 6 is for carrying freezing target cell to carry out refrigeration operation, its body internal surface is the laminated structure of cambered surface, the inner surface of described carrier body is provided with super hydrophobic surface functional area and water-wetted surface functional area, super hydrophobic surface functional area described in described water-wetted surface functional area four periderm around, described water-wetted surface functional area is provided with drop positioning mark.This spline structure freezing/recovery carrier 6 is more conducive to transfer and the location of cell drop, thus be beneficial to cell drop freezing/resuscitation.About the concrete structure of described carrier, can be described in detail by composition graphs 2 below.Wherein,
In the present embodiment, described carrier mechanical action device 4 and described drop change liquid operating means 3 be connected with described central controller respectively 9 with receive corresponding freezing/recovery control instruction.
Described carrier mechanical action device 4 comprises basic machine 401 and the carrier holding action device 41 that is arranged on basic machine 401 and drop pick-up unit 42 (Fig. 4).
Described drop pick-up unit 42 for according to freezing/recovery control instruction corresponding target cell drop adsorbed and transfer to described freezing/ad-hoc location of recovery carrier 6/by target cell drop from described freezing/recovery carrier adsorbs and shifts away.
Described carrier holding action device 41 for according to freezing/recovery control instruction, by described freezing/recovery carrier on complete drop change liquid operation after target cell drop together with described freezing/recovery carrier 6 be moved in described refrigerant container 8 with complete freezing/be moved out to completing freezing target cell drop in described refrigerant container 8 on described operating desk 7 from described refrigerant container 8 together with freezing/recovery carrier 6.
Described drop change liquid operating means 3 for according to freezing/recovery control instruction on described operating desk 7 carrying freezing/recovery carrier 6 on target cell drop carry out drop change liquid operation.
Below, composition graphs 1 ~ 5, further describes concrete formation and the course of work of the automatic pilot of the active somatic cell glass freezing/recovery of the embodiment of the present invention.
Active somatic cell glass freezing/recovery the carrier 6 of the present embodiment as shown in Figure 2, the body 60 of this freezing/recovery carrier 6 for inner surface be the laminated structure of cambered surface.Body 60 inner surface of this freezing/recovery carrier 6 is provided with super hydrophobic surface functional area 61 and water-wetted surface functional area 62, super hydrophobic surface functional area 61 described in described water-wetted surface functional area 62 4 periderm around, and described water-wetted surface functional area 62 is provided with drop positioning mark 600 (location to help cell drop).
In the present embodiment, described water-wetted surface functional area 62 is border circular areas.And described super hydrophobic surface functional area 61 is the circular annular region around described water-wetted surface functional area 62.And the central point of this water-wetted surface functional area 62 overlaps with the minimum point of carrier body 60.Thus be more conducive to the location of cell drop.
Wherein, described super hydrophobic surface functional area 61 is on the inner surface of described carrier, laid the super hydrophobic surface layer of one deck super hydrophobic material or described super hydrophobic surface functional area is by the super hydrophobic surface layer of the subregion of the inner surface of described carrier gained after super-hydrophobic process.
Same, described water-wetted surface functional area 62 is on the inner surface of described carrier, laid the hydrophilic surface layer of one deck water wetted material or described water-wetted surface functional area is by the hydrophilic surface layer of the subregion of the inner surface of described carrier gained after hydrophilic treated.
When adopting the active somatic cell glass freezing/recovery carrier 6 of the present embodiment as when carrying cell drop to carry out cell glass freezing/resuscitation, the combination of water-wetted surface functional area 62 and super hydrophobic surface functional area 61 can make the drop automatic absorbing of specified volume be fixed on water-wetted surface functional area 62, under gravity environment, the cell that the carrier of regular globoidal structure can make drop comprise is positioned on the intersection of cambered surface and objective table automatically.Body surface due to freezing/recovery carrier 6 is cambered surface, and the minimum point of this cambered surface and the point coincides for the water-wetted surface functional area 62 of positioning cells droplet position, thus be beneficial to the automatic location of cell drop.In addition, owing to being super hydrophobic surface functional area 61 around the region of water-wetted surface functional area 62, cell drop can not stick to super hydrophobic surface functional area 61 on the surface, thus further impels cell sap to drip to navigate to and be provided with on the water-wetted surface functional area 62 of drop positioning mark.
Fig. 3 shows the structural representation of the operating desk of the automatic pilot of a kind of active somatic cell glass freezing/recovery in the embodiment of the present invention.This operating desk 7 is surfaces is a platform of plane, and on the surface or transparent operation region 71.This transparent operation region 71 for carry above-mentioned freezing/recovery carrier 6.The operating desk 7 of the present embodiment is preferably movable operating 7, and it connects by arranging the movement that moving-member realizes whole operating desk 7.And the moving-member that operating desk 7 connects is connected with central controller 9, by the movement of central controller 9 control realization operating desk 7, thus can according to the instruction control operation platform 7 of central controller 9 move to specific position with carry out cell freezing/recovery change liquid operation.
Fig. 4 is the structural representation of the carrier mechanical action device of the automatic pilot of a kind of active somatic cell glass freezing/recovery in the embodiment of the present invention.This carrier mechanical action device 4 specifically comprises basic machine 401 and the carrier holding action device 41 that is arranged on basic machine 401 and drop pick-up unit 42.
Described drop pick-up unit 42 comprises detachable drop and picks up pipe 421 and pick up pipe 421 with described detachable drop and be communicated with and control its detachable negative-pressure ward equipment 422 adsorbed.Described drop pick-up unit 42 is fixed on described basic machine 401 by described detachable negative-pressure ward equipment 422.Described detachable negative-pressure ward equipment 422 according to receive freezing/recovery control instruction, corresponding control described detachable drop pick up pipe 421 adsorbed target cell drop and transfer to described freezing/ad-hoc location of recovery carrier 6 on/by target cell drop from described freezing/recovery carrier 6 adsorbs and shifts away (such as, transferring in culture carrier or other containers).
Described carrier holding action device 41 comprises the carrier hold assembly 411 for clamping freezing/recovery carrier and described carrier hold assembly 411 is fixed on the fixed support 412 on described basic machine 401.Described carrier hold assembly 411 according to freezing/recovery control instruction, by described freezing/recovery carrier 6 on complete drop change liquid operation after target cell drop together with described freezing/recovery carrier 6 clamp and be moved in refrigerant container 8 with complete freezing/to press from both sides out completing freezing target cell drop in described refrigerant container 8 from described refrigerant container 8 together with freezing/recovery carrier 6 and to move on on described operating desk 7.
Preferably, the described carrier mechanical action device 4 of the present embodiment is packaged type carrier mechanical action device 4, described carrier mechanical action device 4 also comprises the moving-member 43 be connected with described basic machine, and described carrier mechanical action device 4 can be moved by described moving-member 43 on motion guide rail 404.The moving-member 43 that described carrier mechanical action device 4 connects is connected with central controller 9, by the movement of central controller 9 control realization carrier mechanical action device 4, thus specific position can be moved to carry out the operations such as transfer to cell drop or freezing/recovery carrier 6 according to the instruction control carrier mechanical action device 4 of central controller 9.
Fig. 5 is the structural representation that the drop of the automatic pilot of a kind of active somatic cell glass freezing/recovery in the embodiment of the present invention changes liquid operating means 3.Drop changes that liquid operating means 3 comprises waste liquid eject control 301, culture fluid adds controller 302, drop output pipe 303 and drop intake line 304, and described drop output pipe 303 and two terminals of drop intake line 304 are all connected droplet manipulation pin 305 to draw/to export drop.Described waste liquid eject control 301 is communicated with described drop output pipe 303, and is realized the waste liquid flow whether being extracted out by the waste liquid in the cell drop on freezing/recovery carrier 6 and control to extract out by the On/Off that controls described drop output pipe 303 and the size that controls to open.And described culture fluid is added controller 302 with described drop intake line 304 and is communicated with; and by the On/Off of described drop intake line 304 and the size that controls to open realize whether culture fluid (such as, cryoprotector/recovery liquid) being delivered to described freezing/recovery carrier 6 on cell drop in and control the culture fluid flow of conveying.
Described central controller 9 is added controller 302 with described waste liquid eject control 301 and described culture fluid respectively and is connected, and adds controller 302 work to control described waste liquid eject control 301 and described culture fluid.Concrete, described central controller 9 is by controlling described waste liquid eject control 301, the size realizing the On/Off and control unlatching controlling described drop output pipe 303 realizes the waste liquid flow whether the waste liquid extraction in the cell drop on freezing/recovery carrier 6 and control extracted out, and add controller 302 by controlling described culture fluid, by culture fluid (such as whether the On/Off controlling described drop intake line 304 and the size controlling to open realize, cryoprotector/recovery liquid) be delivered on described culture carrier cell drop in and control the culture fluid flow of conveying.In the present embodiment, described waste liquid eject control 301 and described culture fluid add controller 302 for peristaltic pump or stepper motor.
Preferably, change in liquid operating means 3 at the drop of the present embodiment, the droplet manipulation pin 305 that described drop output pipe 303 is connected with two terminals of drop intake line 304 is dismountable droplet manipulation pin, therefore, the drop of the present embodiment changes liquid operating means and also comprises droplet manipulation pin and change action equipment 306.This droplet manipulation pin is changed action equipment 306 and is connected with described central controller 9, to receive control instruction.This droplet manipulation pin is changed action equipment 306 and is automatically changed for the droplet manipulation pin be connected with two terminals of drop intake line 302 described drop output pipe 301.Concrete, before needing to change the cell drop on freezing/recovery carrier 6 liquid operation or change after liquid operated at every turn, described central controller 9 changes action equipment 306 sending controling instruction to droplet manipulation pin, indicates this droplet manipulation pin replacing action equipment 306 to carry out droplet manipulation pin 305 to corresponding drop output pipe 303 and/or drop intake line 304 and changes.
Preferably, the drop of the present embodiment changes liquid operating means 3 and also comprises the culture fluid be connected with described central controller 9 and choose action equipment 307.This culture fluid choose action equipment 307 for the instruction that receives described central controller 9 to move in described drop intake line 304 to the container 310 filling different culture fluid with obtain corresponding freezing/recovery liquid.The drop of the present embodiment change liquid operating means also comprise for collect via described drop output pipe 303 extract out freezing/recovery carrier 6 on cell drop in the waste liquid collection vessel 311 of waste liquid.
Referring back to Fig. 1, the refrigerant container 8 of the present embodiment built with refrigerant be liquid nitrogen.
In addition, the automatic pilot of the active somatic cell glass freezing/recovery of the present embodiment also comprises the image acquiring device 308 and display 10 that are connected with described central controller 9 respectively.Described image acquiring device 308 for obtain described freezing/recovery carrier 6 on cell drop in cell image information, and after sending to described central controller 9 to process the cell image information got, to be shown by described display 10.Therefore, the image information that can be obtained by this image acquiring device 308 identifies the drop positioning mark on the water-wetted surface functional area 62 of freezing/recovery carrier 6, thus the position that positioning cells drop is placed.
Concrete, described central controller 9 processes (such as to the cell image information that described image acquiring device 308 sends over, the image information of each mark position obtained by image acquiring device 308 successively saves as the image collection of ad-hoc location), to obtain cell image information more clearly, and the cell image information after process is shown by described display 10.Patient can have access to the image collection of any mark position at any time by this display 10, judge the developmental condition of cell and record.In the present embodiment, described image acquiring device 308 is consisted of jointly described fibre-optical cold light source equipment and cell image acquisition equipment CCD.Wherein, fibre-optical cold light source equipment is located at directly over operating desk 7, and just on the transparent operation region 71 of operating desk 7 freezing/recovery carrier 6 on cell drop, and described cell image obtains equipment CCD and is located at immediately below operating desk 7, and just to described fibre-optical cold light source equipment.On the transparent operation region 71 of described light source 20 pairs of operating desks 7 freezing/recovery carrier 6 on cell drop throw light on, and be imaged on and be located at described cell image and obtain on equipment CCD.Understandable, the image acquiring device 308 of the present embodiment also can be incorporated into described drop and change in liquid operating means 3, changes liquid operating means 3 be structure as a whole with described drop.
In addition, the automatic pilot of the active somatic cell glass freezing/recovery of the present embodiment can be built in an active somatic cell incubator, this active somatic cell incubator is built-in with environmental management device to adjust temperature, humidity, pressure and the partial pressure in incubator, thus ensure to meet cell culture environment requirement, make the cell drop on freezing/recovery carrier 6 can not have influence on the process of cell freezing/recovery because changing liquid.
Below, the operating process of the automatic pilot of the active somatic cell glass freezing/recovery of the present embodiment is described in detail:
When sending by central controller 9 the multiple control instruction entering cell freezing pattern, first the built-in environmental management device of active somatic cell incubator can receive the control instruction that central controller 9 sends, and the environment of corresponding adjustment incubator is to the state of compound cells refrigeration operation.Then, carrier mechanical action device 4, according to the control instruction received, is moved to applicable position (making the detachable drop of drop pick-up unit 42 pick up pipe 421 just to target cell drop) by the moving-member 43 of carrier mechanical action device 4.Then, drop pick-up unit 42 is according to the control instruction received, control described detachable drop and pick up pipe 421 adsorbed target cell drop (such as, from culture carrier) and transfer on described operating desk 7 freezing/ad-hoc location (that is, being provided with the water-wetted surface functional area 62 of drop positioning mark) of recovery carrier 6 upper (especially by image acquiring device 308 to locate this ad-hoc location).Then, the moving-member of this operating desk 7 is according to the control instruction received, the operating desk 7 being loaded with freezing/recovery carrier 6 (and cell drop) is moved to drop change between liquid operating means operating space (especially by image acquiring device 308 to locate the drop positioning mark on freezing/recovery carrier 6, the cell sap that movable operating platform 7 makes it carry is dropped in drop and changes on liquid operating means operating point), then, drop changes liquid operating means 3 according to the control instruction received, to on described operating desk 7 freezing/ad-hoc location of recovery carrier 6 on cell drop carry out cell freezing change liquid operation, and change drop composition (such as according to preset program, cryoprotector).Now, record cell image information is obtained by image acquiring device 308.Until described freezing/recovery carrier 6 on target cell reach and reach specified value scope (when namely the cell surface area exposed in the environment reaches preset value) with the poised state of cryoprotector and drop volume, the moving-member 43 of carrier mechanical action device 4 is according to the control instruction received, carrier mechanical action device 4 is moved to applicable position (making the carrier hold assembly 411 of carrier holding action device 41 just to target cell drop), described carrier holding action device 41 by described freezing/recovery carrier 6 on complete drop change the target cell drop after liquid operation together with described freezing/recovery carrier 6 clamps and is moved rapidly into refrigerant container 8 (such as, liquid nitrogen) in freezing to complete.
When sending by central controller 9 the multiple control instruction entering cell recovery pattern, first the built-in environmental management device of active somatic cell incubator can receive the control instruction that central controller 9 sends, and the environment of corresponding adjustment incubator is to the state of compound cells resuscitation.Then, the moving-member 43 of carrier mechanical action device 4 is according to the control instruction received, carrier mechanical action device 4 is moved to applicable position (making the carrier hold assembly 411 of carrier holding action device 41 just to target cell drop), then, carrier holding action device 41, according to the control instruction received, controls described carrier hold assembly 411 and to press from both sides out completing freezing target cell drop in described refrigerant container 8 from described refrigerant container 8 together with freezing/recovery carrier 6 and to move on on described operating desk 7.Then, the moving-member of this operating desk 7 is according to the control instruction received, the operating desk 7 being loaded with freezing/recovery carrier 6 (and cell drop) is moved to drop change between liquid operating means operating space (especially by image acquiring device 308 to locate the drop positioning mark on freezing/recovery carrier 6, the cell sap that movable operating platform 7 makes it carry is dropped in drop and changes on liquid operating means operating point), then, drop changes liquid operating means 3 according to the control instruction received, to on described operating desk 7 freezing/ad-hoc location of recovery carrier 6 on cell drop carry out cell recovery change liquid operation, and change liquid capacity and composition according to preset program.Now, record cell image information is obtained by image acquiring device 308.Until cell reaches the culture fluid poised state of regulation, complete recovery.The moving-member 43 of carrier mechanical action device 4 is according to the control instruction received, carrier mechanical action device 4 is moved to applicable position (making the detachable drop of drop pick-up unit 42 pick up pipe 421 just to target cell drop), described detachable drop picks up pipe 421 according to the control instruction received, by described freezing/recovery carrier 6 on complete drop change liquid operation after target cell drop adsorb and shift away (such as, transferring in culture carrier or other containers).
The automatic pilot of the active somatic cell glass freezing/recovery of the present embodiment, by adopting inner surface to be that the carrier of the laminated structure of cambered surface is as freezing/recovery carrier, and the inner surface of this freezing/recovery carrier is provided with super hydrophobic surface functional area and water-wetted surface functional area, super hydrophobic surface functional area described in described water-wetted surface functional area four periderm around, described water-wetted surface functional area is provided with drop positioning mark, like this, whole freezing/recovery automatic operation process in, by to the identification of drop positioning mark and location, can automatically complete freezing target cell whole freezing/resuscitation process, without the need to artificial participation, increase work efficiency greatly, ensure the stability of operation, promptness and safety.
With reference to figure 6, embodiments provide a kind of method of automatically operating of active somatic cell glass freezing, as shown in Figure 6, the method comprises:
Step S101, sent the multiple control instructions entering cell freezing pattern to multiple actuating unit by central controller;
Step S102, by drop pick-up unit and according to the control instruction that receives, on the ad-hoc location of the freezing carrier that target cell drop absorption migration is carried to operating desk;
Step S103, to be changed liquid operating means by drop and according to the control instruction received, drop is carried out to the target cell drop on the ad-hoc location of the freezing carrier that described operating desk carries and changes liquid operation, and change drop composition according to preset program, reach the cryoprotector poised state of regulation to target cell, and drop volume reaches specified value scope; And
Step S104, by carrier holding action device and according to the control instruction that receives, by described freezing carrier complete drop change liquid operation after target cell drop be moved in described refrigerant container freezing to complete together with described freezing carrier.
Wherein, the structure of the freezing carrier in the present embodiment adopts the structure of the active somatic cell glass freezing/recovery carrier shown in Fig. 1.
The present invention uses the method for automatically operating of the active somatic cell glass freezing shown in Fig. 6 to replace manual work, can increase work efficiency greatly, ensures the stability of operation, promptness and safety.
With reference to figure 7, be the flow chart of the method for automatically operating of a kind of active somatic cell rapid glassization recovery in the embodiment of the present invention, the method comprises:
Step S201, sent the multiple control instructions entering cell recovery pattern to multiple actuating unit by central controller;
Step S202, according to the control instruction that receives, be moved out on operating desk together with freezing carrier by completing freezing target cell drop in refrigerant container from described refrigerant container by carrier holding action device; Wherein, described target cell drop is placed on the ad-hoc location of described freezing carrier;
Step S203, to be changed liquid operating means by drop and according to the control instruction received, drop is carried out to the target cell drop on the ad-hoc location of the freezing carrier that described operating desk carries and changes liquid operation, and change liquid capacity and composition according to preset program, until cell reaches the culture fluid poised state of regulation, complete recovery; And
Step S204, according to the control instruction that receives by drop pick-up unit, described target cell drop is adsorbed from described freezing carrier and shifts away.
Same, the structure of the recovery carrier in the present embodiment adopts the structure of the active somatic cell glass freezing/recovery carrier shown in Fig. 1.
Fig. 6 or Fig. 7 is the flow chart of the method for automatically operating of the active somatic cell glass freezing/recovery exemplified according to the invention process, it must be appreciated, the method of automatically operating step of active somatic cell glass freezing/recovery proposed by the invention is not limited to the execution sequence shown in Fig. 6 or Fig. 7, and those skilled in the art can change arbitrarily the freezing method of automatically operating step of active somatic cell rapid glassization according to spirit of the present invention.
The above is the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications are also considered as protection scope of the present invention.
More than in conjunction with most preferred embodiment, invention has been described, but the present invention is not limited to the embodiment of above announcement, and should contain various carry out according to essence of the present invention amendment, equivalent combinations.
Claims (15)
1. active somatic cell glass freezing/recovery carrier, it is characterized in that, the transparent sheet-like structure of described carrier body to be inner surface be cambered surface, the inner surface of described carrier body is provided with super hydrophobic surface functional area and water-wetted surface functional area, super hydrophobic surface functional area described in described water-wetted surface functional area four periderm around, described water-wetted surface functional area is provided with drop positioning mark.
2. active somatic cell glass freezing/recovery carrier as claimed in claim 1, it is characterized in that, described super hydrophobic surface functional area is circular annular region, and described water-wetted surface functional area is border circular areas.
3. active somatic cell glass freezing/recovery carrier as claimed in claim 2, it is characterized in that, the central point of described water-wetted surface functional area overlaps with the minimum point of described carrier body.
4. active somatic cell glass freezing/recovery carrier as claimed in claim 1, it is characterized in that, described super hydrophobic surface functional area is on the inner surface of described carrier, laid the super hydrophobic surface layer of one deck super hydrophobic material or described super hydrophobic surface functional area is by the super hydrophobic surface layer of the subregion of the inner surface of described carrier gained after super-hydrophobic process;
Described water-wetted surface functional area is on the inner surface of described carrier, laid the hydrophilic surface layer of one deck water wetted material or described water-wetted surface functional area is by the hydrophilic surface layer of the subregion of the inner surface of described carrier gained after hydrophilic treated.
5. the automatic pilot of active somatic cell glass freezing/recovery, it is characterized in that, comprise central controller, carrier mechanical action device, drop change liquid operating means, freezing/recovery carrier, carry described freezing/operating desk of recovery carrier and refrigerant container, wherein
Described carrier mechanical action device and described drop change liquid operating means be connected with described central controller respectively to receive corresponding freezing/recovery control instruction;
Described carrier mechanical action device comprises basic machine and the carrier holding action device that is arranged on basic machine and drop pick-up unit; Described drop pick-up unit for according to freezing/recovery control instruction corresponding target cell drop adsorbed and transfer to described freezing/ad-hoc location of recovery carrier/by target cell drop from described freezing/recovery carrier adsorbs and shifts away; Described carrier holding action device be used for according to freezing/recovery control instruction, by described freezing/recovery carrier on complete drop change liquid operation after target cell drop together with described freezing/recovery carrier be moved in described refrigerant container with complete freezing/be moved out on described operating desk together with freezing/recovery carrier by completing freezing target cell drop in described refrigerant container from described refrigerant container;
Described drop change liquid operating means for according to freezing/recovery control instruction, described operating desk is carried freezing/recovery carrier on target cell drop carry out drop change liquid operation.
6. the automatic pilot of active somatic cell glass freezing/recovery as claimed in claim 5, is characterized in that, described freezing/the active somatic cell glass freezing/recovery carrier of recovery carrier according to any one of Claims 1 to 4; Described ad-hoc location for described in be provided with on the water-wetted surface functional area of drop positioning mark.
7. the automatic pilot of active somatic cell glass freezing/recovery as claimed in claim 5, is characterized in that, also comprises the image acquiring device and display that are connected with described central controller respectively;
Described image acquiring device for obtain described freezing/recovery carrier on cell drop in cell image information, and after the cell image information got is sent to described central controller process, to be shown by described display.
8. the automatic pilot of active somatic cell glass freezing/recovery as claimed in claim 7, is characterized in that, described image acquiring device can change liquid operating means with described drop and be structure as a whole.
9. the automatic pilot of active somatic cell glass freezing/recovery as claimed in claim 5, it is characterized in that, described carrier mechanical action device is packaged type carrier mechanical action device, described carrier mechanical action device also comprises the moving-member be connected with described basic machine, and described carrier mechanical action device can be moved by described moving-member on motion guide rail.
10. the automatic pilot of active somatic cell glass freezing/recovery as claimed in claim 5, it is characterized in that, the fixed support that described carrier holding action device comprises the carrier hold assembly for clamping freezing/recovery carrier and is fixed on by described carrier hold assembly on described basic machine.
The automatic pilot of 11. active somatic cell glass freezing/recoveries as claimed in claim 5, it is characterized in that, described drop pick-up unit comprises detachable drop and picks up pipe and pick up pipe with described detachable drop and be communicated with and control its detachable negative-pressure ward equipment adsorbed.
The automatic pilot of 12. active somatic cell glass freezing/recoveries as claimed in claim 5, it is characterized in that, the refrigerant that described refrigerant container content has is liquid nitrogen.
The automatic pilot of 13. active somatic cell glass freezing/recoveries as claimed in claim 5, it is characterized in that, described automatic operation system is placed in the controlled cell culture incubator of environment.
The method of automatically operating of 14. 1 kinds of active somatic cell glass freezings, is characterized in that, comprises step:
S1, sent the multiple control instructions entering cell freezing pattern to multiple actuating unit by central controller;
S2, by drop pick-up unit and according to the control instruction that receives, on the ad-hoc location of the freezing carrier that target cell drop absorption migration is carried to operating desk;
S3, to be changed liquid operating means by drop and according to the control instruction received, drop is carried out to the target cell drop on the ad-hoc location of the freezing carrier that described operating desk carries and changes liquid operation, until the target cell on described freezing carrier reaches reach specified value scope with the poised state of cryoprotector and drop volume; And
S4, by carrier holding action device and according to the control instruction that receives, by described freezing carrier complete drop change liquid operation after target cell drop be moved in described refrigerant container freezing to complete together with described freezing carrier.
The method of automatically operating of 15. 1 kinds of active somatic cell glass recoveries, is characterized in that, comprise step:
S1, sent the multiple control instructions entering cell recovery pattern to multiple actuating unit by central controller;
S2, according to the control instruction that receives, be moved out on operating desk together with freezing carrier by completing freezing target cell drop in refrigerant container from described refrigerant container by carrier holding action device; Wherein, described target cell drop is placed on the ad-hoc location of described freezing carrier;
S3, to be changed liquid operating means by drop and according to the control instruction received, according to preset program, drop is carried out to the target cell drop on the ad-hoc location of the freezing carrier that described operating desk carries and changes liquid operation, until the target cell on described freezing carrier reaches culture fluid poised state, complete recovery; And
S4, according to the control instruction that receives by drop pick-up unit, described target cell drop is adsorbed from described freezing carrier and shifts away.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410626064.2A CN104396942B (en) | 2014-11-06 | 2014-11-06 | Active somatic cell glass freezing/the method for automatically operating of recovery, system and carrier |
| PCT/CN2014/090998 WO2016070454A1 (en) | 2014-11-06 | 2014-11-13 | Automation device, carrier and method for oocyte in vitro fertilization and cleavage culture |
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| CN201410626064.2A CN104396942B (en) | 2014-11-06 | 2014-11-06 | Active somatic cell glass freezing/the method for automatically operating of recovery, system and carrier |
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| CN112471136A (en) * | 2020-12-04 | 2021-03-12 | 深圳先进技术研究院 | Cell vitrification freezing treatment system and method |
| US11122796B1 (en) | 2016-02-19 | 2021-09-21 | Regents Of The University Of Minnesota | Cryoprotection compositions and methods |
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| WO2017000419A1 (en) * | 2015-06-30 | 2017-01-05 | 徐小杨 | Cell droplet rapid vitrification freeze-thawing carrier and method |
| WO2017041340A1 (en) * | 2015-09-08 | 2017-03-16 | 徐小杨 | Sperm selection method and carrier |
| CN105132364A (en) * | 2015-09-08 | 2015-12-09 | 徐小杨 | Sperm selecting method and carrier |
| CN105132364B (en) * | 2015-09-08 | 2018-12-14 | 徐小杨 | sperm selection method and carrier |
| US11122796B1 (en) | 2016-02-19 | 2021-09-21 | Regents Of The University Of Minnesota | Cryoprotection compositions and methods |
| US10973225B2 (en) * | 2016-10-13 | 2021-04-13 | Shenzhen Vitavitro BiotechCo., Ltd | Vitrification freezing treatment device for cells and treatment method thereof |
| WO2018068339A1 (en) * | 2016-10-13 | 2018-04-19 | 深圳韦拓生物科技有限公司 | Cell vitrification cryopreservation device and method therefor |
| US20190116785A1 (en) * | 2016-10-13 | 2019-04-25 | Shenzhen Vitavitro Biotech Co.,Ltd. | Vitrification freezing treatment device for cells and treatment method thereof |
| CN108270965A (en) * | 2016-12-27 | 2018-07-10 | 奥林巴斯株式会社 | Photographic device, experimental system and its control method and recording medium of experiment |
| US11174094B2 (en) | 2018-02-13 | 2021-11-16 | Boe Technology Group Co., Ltd. | Biological sheet storage device |
| US11702247B2 (en) | 2018-02-13 | 2023-07-18 | Boe Technology Group Co., Ltd. | Biological sheet storage device |
| CN112471136A (en) * | 2020-12-04 | 2021-03-12 | 深圳先进技术研究院 | Cell vitrification freezing treatment system and method |
| WO2022161195A1 (en) * | 2021-02-01 | 2022-08-04 | 深圳拜尔洛克生物技术有限公司 | Thaw recovery system and method for biomaterial |
| CN114020077A (en) * | 2021-11-02 | 2022-02-08 | 上海理工大学 | Cryoprotectant loading device, control system and method for cell tissue sample |
| CN114020077B (en) * | 2021-11-02 | 2022-08-09 | 上海理工大学 | Cryoprotectant loading apparatus and method for tissue samples |
| CN114540183A (en) * | 2022-02-23 | 2022-05-27 | 苏州摩畅科技有限公司 | Automatic liquid changing device |
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