CN114540183A - automatic fluid changer - Google Patents

Abstract

The invention relates to an automatic liquid changing device for supplying liquid to and recovering liquid from a carrier rod carrying active cells before vitrification freezing and/or during rewarming thawing, comprising: a plurality of liquid storage containers; the multi-channel switching valve can communicate each liquid storage container with a liquid changing channel communicated to the carrying rod through a corresponding liquid supply channel according to selection, or communicate the liquid changing channel with a waste liquid channel used for discharging liquid out of the automatic liquid changing device; a pumping device for providing a positive pressure for supplying liquid to the carrier bar and a negative pressure for recovering liquid from the carrier bar; and the control module controls the multi-channel switching valve to alternately communicate the liquid changing channel with each liquid supply channel so that liquid in the liquid storage container is supplied to the carrying rod under the action of the pumping device, and controls the multi-channel switching valve to communicate the liquid changing channel with the waste liquid channel after a corresponding preset time period so as to recover the liquid from the carrying rod under the action of the pumping device.

Description

automatic fluid changer

technical field

The present invention relates to an automatic liquid exchange device for supplying and recovering liquid to the surrounding environment of active cells before vitrification and/or during rewarming and thawing.

Background technique

Viable cells need to be vitrified during storage to maintain their viability, and need to be rewarmed and thawed when used. Before vitrification, the environment of active cells needs to be exchanged, and the cell culture medium needs to be replaced with vitrification solution. After rewarming and thawing, the vitrification solution should be replaced with cell culture medium. During vitrification, rewarming and thawing, active cells need to be immersed in a variety of specific liquids in a certain order to gradually change the external environment of the cells through the diffusion of liquids, so that cells can adapt to the slow changes during the liquid exchange process. the external liquid environment to remain active.

The current mainstream medium exchange method is still the manual operation of embryologists. Taking the Kitazato method currently used in hospitals as an example, the standard operation process is to suck and push cells through a pipette, and cells are transferred from cell culture medium to cell culture medium one by one. Several equilibrated solutions were held for a certain period of time, and finally transferred to a vitrified freezing solution. After the environment surrounding the viable cells has been replaced with a vitrification solution, the viable cells can be snap frozen in liquid nitrogen at a rate of, for example, -23000°C/min. When the viable cells need to be used, the viable cells are rewarmed and thawed. First, suck the active cells with a pipette, put them in the thawing solution at 37 °C for 1 minute, then take out the active cells from the thawing solution, and transfer them to the dilution solution and washing solution for a certain period of time. The cells are ready for use.

For example, for oocytes, a standard procedure is to place the oocytes in a petri dish, soak them in 20 μL of the base solution (cell culture medium), then add 20 μL of the equilibration solution to the dish, and after 3 minutes Add 20μL of equilibration solution, then add 240μL of equilibration solution for 3 minutes, after 9 minutes, pipette the oocytes and put them into 300μL of vitrification solution for 0.5 minutes, then pipette the oocytes and put them into another 300μL Stay in the vitrification solution for 0.5 minutes. When rewarming and thawing, first suck the oocytes with a pipette and put them into the thawing solution at 37°C. After 1 minute, use a pipette to take out the oocytes from the above thawing solution, put them in 300 μL of dilution solution for 3 minutes, and then Place in wash solution for 5 minutes, then in another wash solution for 1 minute before removing the oocytes and placing them in a vessel for further use.

For embryonic cells, the standard operating procedure is, for example: place the embryonic cells in a petri dish, soak them in 300μL of equilibration solution for 12-15 minutes, then pipette the embryonic cells and place them in 300μL of vitrification solution for 0.5 minutes, and then pipette the embryonic cells into another 300 μL of vitrification solution for 0.5 minutes. When rewarming and thawing, first suck the embryonic cells with a pipette and put them into the thawing solution at 37°C. After 1 minute, use a pipette to remove the embryonic cells from the above thawing solution, put them in 300 μL of dilution solution for 3 minutes, and then put them into the thawing solution. 300 μL of wash solution for 5 minutes, then 300 μL of another wash solution for 1 minute, and then the embryonic cells were removed and placed in a vessel for further use.

The disadvantage of the prior art is that manual medium change requires an experienced embryologist to operate, which requires 2-3 years of training and operating experience, high labor costs, and due to the size of the active cells, the medium change process is highly dependent on the embryologist. Feel, once the error will cause damage to the active cells.

Since the operation time during vitrification and rewarming of viable cells is very short (for example, as mentioned above, the pipette takes only about 1 minute after the viable cells are placed in the thawing solution), manual can be done faster and more Quickly, and directly putting the cells into the thawing solution using the carrier rod that takes out the active cells from the liquid nitrogen that preserves the active cells at low temperature is also in line with the operating habits of operators such as doctors. Therefore, for the carrier rod where the active cells are placed manually. There is no urgent need to automate the process of entering liquid nitrogen for vitrification and cryopreservation on the carrier rod, and removing the carrier rod and placing it in a thawing solution to thaw viable cells. However, for the liquid changing process before and after vitrification, freezing and rewarming and thawing mentioned in the above standard operating procedures, due to the many steps, the variety and quantity of liquid used, the residence time required for each step is also different, so manual operation and Not the best choice.

In this regard, some automatic liquid exchange devices and methods have also been proposed in the prior art. The Gavi system from Australia's GeneaBiomedx enables automated media changes before vitrification and after rewarming and thawing. To do this, the cells need to be manually fixed in the cell tank of the special medium exchange container (consumable), and then the equilibration solution, vitrification solution, and cell culture solution are added and removed to the medium exchange container through the three-dimensional mobile platform to control the pipette gun. Liquid, etc., the liquid exchange is realized by the principle of diffusion. The liquid exchange container can be heat-sealed with a heat-sealing film to achieve isolation from the outside world during low-temperature storage. The problem is that the liquid exchange container used as a consumable in this system is completely different from the pipette for transferring cells and the carrier rod for placing in the cryogenic tank for cryogenic freezing, which are routinely used by operators such as doctors. the practice of doctors. In addition, because the movement of the three-dimensional moving platform controlling the pipette gun is complex, the cost of the system is too expensive.

The Sarah system of FertileSafe in Israel realizes automatic liquid exchange and vitrification before vitrification and after rewarming and thawing, but the vitrification process is not fully automated, excluding the heat sealing part. Cryopreservation, rewarming and thawing still require manual operations. The way to automate the liquid exchange before vitrification and after rewarming and thawing is to distribute different solutions (equilibration solution, vitrification solution, cell culture solution, thawing solution, liquid nitrogen, etc.) on a roulette that can rotate horizontally superior. Viable cells are loaded into a special pipette (consumable) mounted on a z-axis movement module that can move in a z-axis direction perpendicular to the surface of the wheel. The roulette transfers the corresponding solution to the right under the pipette by rotating, and the pipette moves up and down along the z-axis direction under the action of the z-axis moving module to immerse and leave different solutions and achieve a predetermined residence time in different solutions. The disadvantage of this system is that various solutions are placed on the roulette for immersion of the pipette containing the active cells. If these solutions are not shared by different active cells, each solution change takes a lot of time, which is not suitable for continuous pipeline operations. , but there is a potential risk of cross-contamination if different active cells share these solutions. In addition, since the system involves the movement of two components, the z-axis moving module and the wheel disc, the system is complicated and the cost is high.

Therefore, it is desirable to provide a device that automates the steps of liquid exchange before vitrification and after rewarming and thawing, while changing the operating habits of operators such as doctors as little as possible. On this basis, it is also hoped that one carrier rod and one reagent can be realized, so as to avoid sharing carrier rods or various reagent solutions for active cells, and avoid possible cross-contamination in the process. Obviously, it is also hoped that the cost of the device can be controlled at a reasonable level.

SUMMARY OF THE INVENTION

In view of the above technical problems, the present invention provides an automatic liquid exchange device for supplying liquid to and recovering liquid from the carrier rod loaded with active cells before vitrification and/or during the rewarming and thawing process, Wherein, the automatic liquid exchange device includes: a plurality of liquid storage containers, the liquid storage containers are used to respectively store various liquids required before vitrification and freezing and/or in the process of rewarming and thawing; a multi-channel switching valve, the The channel switching valve can connect each of the liquid storage containers with the liquid exchange channel connected to the carrier rod through the corresponding liquid supply channel according to the choice, or connect the liquid exchange channel with the liquid exchange channel used for the automatic liquid exchange device. The waste liquid channel that discharges the liquid outside is connected; the pump pressure device is used to provide a positive pressure for supplying the liquid to the load rod and a negative pressure for recovering the liquid from the load rod; a control module, so The control module controls the multi-channel switching valve to connect the liquid exchange channel with each of the liquid supply channels in turn, so that the liquid in the liquid storage container is supplied to the liquid storage container under the action of the pumping device. The carrier rod is controlled, and after a corresponding predetermined period of time, the multi-channel switching valve is controlled to communicate the liquid exchange channel with the waste liquid channel, so as to remove the liquid from the carrier rod under the action of the pumping device. Recycle liquid.

Since the active cells are placed on the carrier rod, the various liquids that need to be immersed are successively replaced by the surrounding liquid under the positive pressure provided by the pumping device, and after staying for a predetermined time under the negative pressure of the pumping device. It is detached from the surrounding environment of the active cells, so the active cells do not need to move during the whole process of changing the medium before vitrification and rewarming and thawing, and there is no mechanical movement that needs to be precisely controlled in the system. On the one hand, the system design is simplified, and on the other hand, the residence time of active cells in various liquids can be controlled more precisely than manual operation or immersion by mechanical motion.

The carrier rod carries active cells, and after the liquid exchange is completed, all liquids used in the process have left the automatic liquid exchange device through the waste liquid channel, so the operation of the new active cells on the next carrier rod has been completely avoided. Possibility of cross contamination.

In addition, because the automatic liquid exchange device only communicates with the carrier rod through the liquid exchange channel, the interface is simple, and no complicated design and change of the carrier rod are required. Necessary adaptive design is carried out on the basis of the rod, so that it can have a simple and consistent structure compared with the existing carrier rod to meet the original operating habits of operators such as doctors. When using the automatic medium exchange device of the present invention, in addition to placing the active cells on the carrier rod with a pipette before the medium exchange, removing the active cells from the carrier rod for subsequent use after the liquid exchange is completed, and in the glass After the liquid change before freezing is completed, put the carrier rod directly into the liquid nitrogen cryogenic tank. Except for the operation steps that are exactly the same as the traditional operation habits, the rest of the time-consuming and experience-required liquid change steps are all suitable for doctors and other operations. Both implement functional encapsulation.

According to a preferred embodiment of the automatic liquid exchange device of the present invention, the liquid stored in the liquid storage container includes all or a part of the following liquids: basic solution, freezing equilibrium solution, vitrification freezing solution, dilution solution, washing solution.

That is to say, the basic solution, freezing equilibration solution, and vitrification freezing solution that need to be added to the surrounding environment of active cells in sequence before vitrification and freezing can be stored in several liquid storage containers of an automatic liquid exchange device according to the present invention. When the carrier rod is communicated with the liquid exchange channel of the automatic liquid exchange device, the liquid exchange before vitrification and freezing can be realized, and the dilution solution and the washing solution are stored in several liquid storage containers of another automatic liquid exchange device according to the present invention. , when the carrier rod is communicated with the liquid exchange channel of the automatic liquid exchange device, the liquid exchange in the process of rewarming and thawing can be realized.

Obviously, any of the above-mentioned base solutions, freezing equilibration solutions, vitrification freezing solutions, diluting solutions, washing solutions are not limited to being placed in only one liquid storage container. Obviously, multiple liquid storage containers can also be arranged in the automatic liquid exchange device to store the same liquid according to needs and usage, so as to appropriately reduce the downtime for liquid exchange.

Obviously, it should also be understood that there may be more than one freezing equilibration solution, vitrification freezing solution, diluting solution, and washing solution here. Equilibrium solutions, multiple dilution solutions, or multiple washing solutions, etc., can also be arranged with multiple liquid storage containers for storing different types of frozen balancing solutions, dilution solutions, or washing solutions, etc. respectively. For example, corresponding to the 20 μL, 20 μL and 240 μL equilibration solutions added to the oocyte environment in the prior art Kitazato method, if three different equilibration solutions are desired to be used in practice, these three The equilibration solution is placed in three different reservoirs of the automatic fluid changer of the present invention.

Preferably, the reservoir container is replaceable or refillable. When the liquid in the liquid storage container is exhausted, the entire liquid storage container can be replaced as a whole, or the liquid storage container can be filled and replenished. In the case of replacing the liquid storage container, it is advantageous to provide the liquid storage container so that it can be quickly plugged in and replaced.

In order to monitor the liquid level in the liquid storage container, a liquid level monitoring device can be provided and an alarm signal can be given if the liquid level of the liquid storage container is below a predetermined low threshold value. In the case where the liquid storage container can be filled, it is advantageous to be able to give an alarm signal by the liquid level monitoring device if the liquid level of the liquid storage container is above a predetermined high threshold value. At this point, the filling of the reservoir can be stopped.

The liquid level monitoring device can also be used to check the liquid level in each liquid storage container before each liquid exchange. An alarm signal is issued to prompt the doctor or operator to fill the corresponding liquid storage container with insufficient liquid level.

The pumping device provides the positive pressure required for supplying the liquid and the negative pressure required for recovering the liquid. The multi-channel switching valve connects the liquid exchange channel with the waste liquid channel or the corresponding liquid supply channel according to the choice, and the combination of the two can already be realized. For liquid supply and recovery. In order to control the amount of liquid exchange more precisely, it is preferable to configure an on-off valve that can be controlled independently for each of the liquid supply channels and the waste liquid channels correspondingly. The connection and disconnection between each liquid supply channel and the liquid exchange channel are independently controlled by an on-off valve corresponding to each liquid supply channel. The connection and disconnection of the waste liquid channel and the liquid exchange channel are independently controlled by the on-off valve corresponding to the waste liquid channel.

The on-off valve is preferably a solenoid valve. However, it is also conceivable to use a hydraulic valve or a pneumatic valve as the on-off valve. In a preferred implementation form of the automatic liquid exchange device of the present invention, each liquid supply channel or waste liquid channel can be correspondingly provided with a separate on-off valve, such as a solenoid valve. The solenoid valve switches on and off the liquid supply channel or the waste liquid channel under the action of the control module. Alternatively, it is also possible to control a plurality of each liquid supply channel or a plurality of waste liquid channels through a multi-way valve, so as to ensure that at most one channel is connected according to the selection at any time. Of course, there is a functional overlap between the multi-way valve and the multi-channel switching valve in series. The primary purpose of setting the multi-way valve here should be to ensure the rapid connection and disconnection of each liquid supply channel or waste liquid channel and the liquid exchange channel. Therefore, The selection of a multi-way valve as an on-off valve should focus on the response speed.

According to a preferred embodiment of the automatic fluid exchange device of the present invention, the multi-channel switching valve is a rotary valve. The rotary valve is selectively rotated to a position connecting the exchange fluid passage with one of the supply fluid passages or the waste fluid passage at any time. Preferably, the rotary valve can also be rotated to the position of disconnecting each liquid supply channel and waste liquid channel, so as to prevent the liquid from accidentally flowing out of the automatic liquid exchange device.

In a preferred embodiment of the automatic liquid exchange device of the present invention, the pumping device includes a positive pressure pump and a negative pressure pump. The positive pressure pump is used to provide positive pressure to the liquid supply channel when the liquid supply channel is communicated with the liquid exchange channel, so as to transfer the liquid in the liquid storage container connected to the corresponding liquid supply channel through the liquid exchange channel. The fluid channel is supplied to the carrier rod. The negative pressure pump is used to provide negative pressure to the waste liquid channel when the waste liquid channel is in communication with the liquid exchange channel, so as to pump the liquid in the load rod back into the waste liquid channel and thereby discharge the automatic liquid exchange device.

The pumping device can continuously provide positive pressure, or can only provide positive pressure when each liquid supply channel is in communication with the liquid exchange channel. In the former case, the on-off valve additionally controls the communication and disconnection of the liquid exchange channel and each liquid supply channel. In either case, once the liquid exchange channel communicates with the corresponding liquid supply channel, the liquid is supplied to the carrier rod through the passage formed by the liquid exchange channel and the corresponding liquid supply channel under the action of the positive pressure provided by the pumping device.

Similarly, the pumping device may also provide negative pressure continuously or only when the waste fluid channel is in communication with the exchange fluid channel. Once the liquid exchange channel is communicated with the waste liquid channel, the liquid in the carrier rod is drawn out of the automatic liquid exchange device through the passage formed by the liquid exchange channel and the waste liquid channel under the action of the negative pressure of the pumping device.

According to a preferred implementation form of the automatic liquid exchange device of the present invention, the on-off valve corresponding to each liquid supply channel can be optionally arranged upstream or downstream of each liquid storage container, as long as it can be switched on and off. The function of opening the liquid passage is enough.

Preferably, the automatic liquid exchange device further includes a waste liquid container. The waste liquid container is communicated with the multi-channel switching valve through the waste liquid channel, and is used for temporarily storing the waste liquid from the waste liquid channel. An on-off valve correspondingly assigned to the waste liquid channel can optionally be arranged upstream or downstream of the waste liquid container.

It will be readily understood that the waste container may be replaceable or liquid-drainable. When the liquid in the waste liquid container reaches a predetermined high threshold level, the entire waste liquid container can be replaced as a whole, or the liquid in the waste liquid container can be directly discharged. In the case of replacing the waste liquid container, it is advantageous to provide the waste liquid container so that it can be quickly plugged in and replaced.

Although the waste liquid can be temporarily stored in the waste liquid container as described above and then selectively discharged or removed from the automatic liquid changer with the replacement of the entire waste liquid container, the waste liquid can obviously be discharged from the automatic liquid changer at any time. Whether to discharge directly through the waste liquid channel, or directly from the waste liquid container if a waste liquid container is provided.

Similarly, each of the above-mentioned liquid storage containers can also be communicated with the corresponding liquid source so as to be automatically filled and replenished from the outside at all times and does not have to be replaced or manually filled and replenished.

In order to precisely control the supply amount of each liquid, the control module controls the corresponding solenoid valve for each liquid supply channel and the corresponding solenoid valve for the waste liquid channel when the multi-channel switching valve is switched, so that each liquid supply channel and waste liquid channel are correspondingly equipped. The liquid channels remain open to prevent mixed flow between the supply channels and between them and the waste channel.

Preferably, each liquid storage container is integrated in a container assembly that can be replaced as a whole. Further preferably, waste liquid containers can also be integrated in this container assembly together with the respective liquid storage containers. In this way, the replacement of the container assembly makes it easy to replace all the reservoir containers at once, as well as the waste container when necessary.

As an alternative or supplement, it can also be considered to integrate the liquid storage container for storing the liquid required for liquid exchange before vitrification and freezing in one of the above-mentioned container components, and the liquid storage container for storing the liquid required for rewarming and thawing liquid exchange. Integrated into the other of the container assemblies described above, the waste container may optionally be integrated into one of the two container assemblies or provided separately.

According to a preferred embodiment of the carrier rod of the present invention, the fluid exchange port is designed to be suitable for a Luer connection and communicate with a corresponding Luer connection of an external automatic fluid exchange device. However, it is also possible to communicate with an external automatic fluid changer via a dedicated carrier clamp. For example, according to another aspect of the present invention, there is provided a carrier rod clamp, which is used for connecting the carrier rod with an external automatic liquid exchange device when clamping the above-mentioned carrier rod, with a base and an opening The base has a accommodating part suitable for accommodating the carrier rod in a manner that the notch of the cell exchange tank is upward, and the opening and closing part is arranged on the base in a manner that can be pivotally opened and closed around the base On the opening and closing part, a pipe joint that penetrates from the side of the opening and closing part toward the liquid exchange port to the side that faces away from the base is fixed, and the pipe joint is opposite to the opening and closing part. One side of the base is suitable for communicating with an external automatic liquid exchange device, and the side of the opening and closing part facing the liquid exchange port is suitable for aligning when the opening and closing part is closed on the base The liquid exchange port of the carrier rod accommodated in the accommodating part, the opening and closing part is arranged with a sealing ring around the pipe joint on the side facing the liquid exchange port, and the sealing ring is suitable for the opening and closing When the part is closed to the base, it is pressed against the liquid exchange port of the carrier rod, so as to realize the sealing of the periphery when the liquid exchange port is connected.

Obviously, the design of the opening and closing portion can be changed according to the position of the liquid exchange port on the carrier rod relative to the cell liquid exchange tank. In the case that both the cell exchange tank and the exchange port are designed on the front of the carrier rod and the opening and closing part will block the cell exchange tank when the opening and closing part is closed on the base, the opening and closing part can be set on the opening and closing part according to the needs. The side facing the liquid exchange port runs through to the side facing away from the base, and the size and position of the cutout are designed so as not to obscure the cell exchange tank on the carrier rod when the opening and closing part is closed to the base notch and bottom. If the liquid exchange port of the carrier rod is not arranged on the front side of the carrier rod together with the cell liquid exchange tank but on the side or back of the carrier rod, or the pivot shaft of the opening and closing part is not arranged on the side close to the cell liquid exchange tank but is arranged On the side away from the cell exchange tank or on the side of the carrier rod, it is not necessary to design the above-mentioned gap on the opening and closing part, because the opening and closing part will not block the notch of the cell exchange tank during the pivotal opening and closing process. and bottom. Not blocking the notch allows the active cells to be easily put into the cell exchange tank, and not blocking the bottom (in the case where the bottom of the cell exchange tank is made of a transparent material) facilitates observation of the cells in the cell exchange tank through a microscope. Active cells without having to remove them.

The advantage of the above-mentioned carrier rod clamp is that the pipe joint is continuously connected to the liquid supply channel of the automatic liquid exchange device on the side facing away from the base, and the pivoting of the opening and closing part and the pressing of the sealing ring together realize quick connection and reliable sealing. Compared with the Luer connection, the carrier pole can be deployed more quickly and the work efficiency is improved.

Description of drawings

The present invention will be described in detail below with reference to the accompanying drawings. In the attached image:

Fig. 1 schematically shows the principle diagram of the automatic liquid exchange device according to the present invention;

Figure 2 schematically shows a perspective view of a carrier rod according to the invention;

Figure 3 schematically shows a perspective view in longitudinal section of a carrier bar according to the invention;

Figure 4 schematically shows an enlarged view of the portion of the carrier rod according to the present invention within the dashed box shown in Figure 3;

Figure 5 schematically shows an assembly drawing of the carrier rod according to the invention arranged on the carrier rod clamp.

Detailed ways

FIG. 1 schematically shows a schematic diagram of an automatic liquid exchange device 110 according to the present invention. The liquid storage containers 3 to 8 for storing liquid and the waste liquid container 11 for storing waste liquid are shown in the dashed box. The automatic liquid exchange device 110 communicates with the carrier rod 100 (not shown in this figure) through the liquid exchange channel 10 . The dashed box indicates that these containers together form a container assembly that can be replaced as a whole.

As shown in FIG. 1 , the positive pressure device 1 in the pumping device provides positive pressure to the liquid storage containers 3 to 8 , and the negative pressure device 13 provides negative pressure to the waste liquid channel 14 . When the multi-channel switching valve 9 communicates the liquid exchange channel 10 with any of the liquid supply channels 23 to 28 , the liquids in the corresponding liquid storage containers 3 to 8 are supplied to the liquid exchange channel 10 and then to the carrier rod. When the multi-channel switching valve 9 communicates the liquid exchange channel 10 with the waste liquid channel 14 , the liquid in the carrier rod is drawn back through the liquid exchange channel 10 and discharged from the automatic liquid exchange device through the waste liquid channel 14 .

Figure 2 schematically shows a perspective view of a carrier bar according to the invention. The overall outline of the carrier rod 100 is an elongated shape, the rear part is used for holding, and the front part is used for carrying active cells. The front part of the carrier rod has a cell exchange tank 101, and the rear part has an information area for marking visually visible or machine-readable information. A liquid exchange port 103 is arranged in the middle part of the carrier rod, preferably in the front middle part close to the cell liquid exchange tank 101 , for communicating with the liquid exchange channel 10 of the automatic liquid exchange device 110 . The top of the cell exchange tank 101, that is, the side facing the front side of the carrier rod 100, has an open notch, which is convenient for doctors or operators to pick up and place active cells with a pipette and other tools. There is a channel 104 communicating with the liquid exchange port 103, as shown in FIG. 3 . As shown in detail in FIG. 4 , the channel 104 has a narrowed portion 105 on the side close to the cell exchange tank 101, so that the liquid flowing under the action of the pumping device can be smoothly supplied into the cell exchange tank 101 or from the cell exchange tank 101. Take out the cell exchange tank 101 .

A cell fixation part 102 is provided in the cell exchange tank 101 , preferably a pit away from the channel 104 as shown in FIG. 3 . In this way, after the cells are put into the cell fixation part 102 in the cell exchange tank 101, they do not move in position with the supply and extraction of the liquid.

The way of fluid communication between the carrier rod 100 and the automatic fluid exchange device 110 is not shown in the drawings. A direct Luer connection is a simple rotation, but the use of a carrier rod clamp 111 shown in Figure 5 is also contemplated, in which the carrier rod 100 is received in the receptacle 116 of the base 113 of the carrier rod clamp 111, and the cells are exchanged. The notch of the slot 101 is upward. The opening and closing portion 112 is arranged on the base 113 in such a manner that it can be pivotally opened and closed around the base 113 . The pipe joint 114 is fixed on the opening and closing part 112 , and penetrates from the side of the opening and closing part 112 toward the liquid exchange port 103 to the side facing away from the base 113 . The liquid exchange device 110 is connected to each other, and the side of the opening and closing portion 112 facing the liquid exchange port 103 is adapted to align with the liquid exchange port of the carrier rod 100 accommodated in the accommodating portion 116 when the opening and closing portion 112 is closed to the base 113 . 103, the opening and closing part 112 is arranged with a sealing ring (not shown) around the pipe joint 114 on the side facing the liquid exchange port 103, and the sealing ring is suitable for pressing against the carrier rod when the opening and closing part 112 is closed to the base 113 100 on the liquid exchange port 103, so as to achieve sealing to the periphery when the liquid exchange port 103 is communicated. The size and position of the opening 115 provided on the opening and closing part 112 from the side facing the liquid exchange port 103 to the side facing away from the base 113 is designed to be closed at the opening and closing part 112 . When on the base 113, the notch and bottom of the cell exchange tank 101 on the carrier rod are not blocked.

Example 1

The oocytes are placed in the cell fixing part 102 in the cell exchange tank 101 of the carrier rod 100 with a pipette, and then the liquid exchange port 103 of the carrier rod 100 is communicated with the liquid exchange channel 10 of the automatic liquid exchange device 110. Rotate the multi-channel switching valve 9, connect the liquid exchange channel 10 with the liquid supply channel 23, and turn on the corresponding switch valve, so that the cell culture solution stored in the liquid storage container 3 flows into the carrier rod in an appropriate amount under the action of the positive pressure device 1. Submerge the oocytes in the cell exchange tank 101 of 100 . Then, firstly rotate the multi-channel switching valve 9, connect the liquid exchange channel 10 with the waste liquid channel 14, and turn on the corresponding switch valve 12, so that the cell exchange tank 101 of the carrier rod 100 is immersed in the cell culture solution of the oocytes at Under the action of the negative pressure device 1, it is drawn out from the carrier rod 100 (as far as possible), and then the multi-channel switching valve 9 is rotated to connect the liquid exchange channel 10 with the liquid supply channel 24, and the corresponding switch valve is turned on, so that the Under the action of the positive pressure device 1, an appropriate amount of the frozen equilibrium solution stored in the liquid storage container 4 flows into the cell exchange tank 101 of the carrier rod 100 to immerse the oocytes and keep for 3 minutes. Then, similarly by rotating the multi-channel switching valve 9 as needed, the freezing equilibration solution and vitrification freezing solution in the corresponding liquid storage container are sequentially supplied to the carrier rod 100 and after a predetermined time (eg, 9 minutes, 0.5 minutes) The prompt information of the completion of the liquid exchange is provided, so that the operator such as the doctor can remove the carrier rod 100 from the automatic liquid exchange device 110, disconnect the liquid communication between the carrier rod 100 and the liquid exchange channel 10 of the automatic liquid exchange device 110, and connect the carrier rod 100 to the liquid exchange channel 10. 100 sets of rod sleeves are then snap frozen in liquid nitrogen at a rate of, for example, -23000°C/min, in a cryogenic tank with their loaded oocytes.

When it is necessary to rewarm and thaw, the operator such as the doctor first takes out the carrier rod 100 carrying oocytes from the cryogenic tank, slides off the rod cover, and then places the cell exchange tank 101 carrying oocytes in the front of the rod cover. After soaking for 1 minute in a container filled with thawing liquid at 37° C., the liquid exchange port 103 is connected to the automatic liquid exchange device 110 . First, turn on the liquid exchange channel 10 and the waste liquid channel 14 by rotating the multi-channel switching valve 9, extract the thawed liquid remaining in the cell liquid exchange tank 101 of the carrier rod 100, and then rotate the multi-channel switching valve 9 to connect the liquid exchange channel 10. and the corresponding liquid supply channel, supply the diluted solution from the corresponding liquid storage container to the cell liquid exchange tank of the carrier rod 100, keep it for 3 minutes, and then turn on the liquid exchange channel 10 and waste liquid by rotating the multi-channel switching valve 9. Channel 14, draw out the diluted solution in the cell exchange tank 101 of the carrier rod 100, then rotate the multi-channel switching valve 9, connect the exchange channel 10 and the corresponding liquid supply channel, and supply the washing solution from the corresponding liquid storage container to the Place the rod 100 in the cell exchange tank for 3 minutes. Then, an operator such as a doctor can use a pipette to take out the oocyte from the cell fixing part 102 in the cell exchange tank 101 of the carrier rod 100 for further use. Before taking out the oocyte with a pipette, an operator such as a doctor can optionally determine whether to extract the residual washing solution in the cell exchange tank 101 of the carrier rod 100 by the automatic liquid exchange device 110 according to the operation habits.

As can be seen from the above, in the whole process of before vitrification, cryogenic freezing and rewarming and thawing, it is not necessary to use a pipette to remove cells from the cell exchange tank 101 of the carrier rod 100 for placing in the cryogenic tank. Before being placed in the cryogenic tank, the notch of the cell exchange tank 101 of the carrier rod 100 can be simply closed by sliding the rod cover. When taken out from the cryogenic tank, the notch of the cell exchange tank 101 of the carrier rod 100 can be exposed again by sliding the rod cover.

Embodiment 2

The embryonic cells are placed in the cell fixing part 102 in the cell exchange tank 101 of the carrier rod 100 with a pipette, and then the liquid exchange port 103 of the carrier rod 100 is communicated with the liquid exchange channel 10 of the automatic liquid exchange device 110. Rotate the multi-channel switching valve 9, connect the liquid exchange channel 10 with the liquid supply channel 23, and turn on the corresponding switch valve, so that the cell culture solution stored in the liquid storage container 3 flows into the carrier rod in an appropriate amount under the action of the positive pressure device 1. Immerse embryonic cells in 100 cell exchange tank 101. Then, firstly rotate the multi-channel switching valve 9 to connect the liquid exchange channel 10 with the waste liquid channel 14, and turn on the corresponding switch valve 12, so that the cell exchange tank 101 of the carrier rod 100 is immersed in the cell culture solution of the embryo cells in the negative Under the action of the pressure device 1, it is drawn (as far as possible) from the carrier rod 100, and then the multi-channel switching valve 9 is rotated to connect the liquid exchange channel 10 with the liquid supply channel 24, and the corresponding switch valve is turned on, so that the storage Under the action of the positive pressure device 1, the frozen equilibrium solution stored in the liquid container 4 flows into the cell exchange tank 101 of the carrier rod 100 in an appropriate amount to immerse the embryonic cells for 12-15 minutes. Then, firstly rotate the multi-channel switching valve 9, connect the liquid exchange channel 10 with the waste liquid channel 14, and turn on the corresponding switch valve 12, so that the cell exchange tank 101 of the load rod 100 is immersed in the frozen equilibrium solution of the embryo cells in the negative state. Under the action of the pressure device 1, it is drawn out of the carrier rod 100 (as far as possible), and then the multi-channel switching valve 9 is rotated to connect the liquid exchange channel 10 with the liquid supply channel 24, and the corresponding switch valve is turned on, so that the corresponding Under the action of the positive pressure device 1, the vitrification solution stored in the liquid storage container flows into the cell exchange tank 101 of the carrier rod 100 in an appropriate amount to immerse the embryonic cells for 0.5 minutes. Then the vitrification solution is drawn out again, and the vitrification solution stored in the corresponding liquid storage container is again supplied to the carrier rod 100 in an appropriate amount under the action of the positive pressure device 1, and kept for 0.5 minutes, and then replaced before the vitrification and freezing. The prompt message of the completion of the liquid is displayed, so that the operator such as the doctor can remove the carrier rod 100 from the automatic liquid exchange device 110, disconnect the liquid communication between the carrier rod 100 and the liquid exchange channel 10 of the automatic liquid exchange device 110, and set the carrier rod 100 The upper rod cover is then snap frozen with liquid nitrogen at a rate of, for example, -23000°C/min, in a cryogenic bath with the embryonic cells it carries.

When rewarming and thawing is required, the doctor and other operators first take out the carrier rod 100 carrying the embryonic cells from the cryogenic tank, slide off the rod cover, and add 37°C thawing solution to the cell exchange tank 101 with a pipette gun. After soaking for 3 minutes, the liquid exchange port 103 is connected to the automatic liquid exchange device 104 . First, turn on the liquid exchange channel 10 and the waste liquid channel 14 by rotating the multi-channel switching valve 9, extract the thawing liquid remaining in the cell exchange liquid tank 101 of the carrier rod 100, and then rotate the multi-channel switching valve 9 to connect the liquid exchange channel 10. and the corresponding liquid supply channel, supply the diluted solution from the corresponding liquid storage container to the cell liquid exchange tank of the carrier rod 100, keep it for 3 minutes, and then turn on the liquid exchange channel 10 and waste liquid by rotating the multi-channel switching valve 9. Channel 14, draw out the diluted solution in the cell exchange tank 101 of the carrier rod 100, then rotate the multi-channel switching valve 9, connect the exchange channel 10 and the corresponding liquid supply channel, and supply the washing solution from the corresponding liquid storage container to the Place the rod 100 in the cell exchange tank for 5 minutes. Then, the washing solution is drawn out again, and another washing solution stored in another corresponding liquid storage container is again supplied to the carrier rod 100 in an appropriate amount under the action of the positive pressure device 1 for 0.5 minutes. At this time, the automatic liquid exchange device or the carrier rod will give a prompt message that the liquid exchange is completed, and operators such as doctors can use a pipette to take out the embryonic cells from the cell fixing part 102 in the cell liquid exchange tank 101 of the carrier rod 100 for further use. Before taking out the embryonic cells with a pipette, an operator such as a doctor may optionally determine whether to extract the residual washing solution in the cell exchange tank 101 of the carrier rod 100 by the automatic liquid exchange device 110 according to the operation habits.

The preferred embodiments of the present invention have been disclosed above, however, the spirit and scope of the present invention are not limited to the specific contents disclosed. Those skilled in the art can develop more embodiments and specific applications through the teaching of the present invention, and these embodiments and specific applications also belong to the spirit and scope of the present invention. Therefore, it can be understood that the specific embodiments of the present invention do not further limit the spirit and scope of the invention defined by the claims.

List of reference signs

100 load bar

101 Cell Exchanger

102 Cell Fixation Section

103 Fluid change port

104 channels

105 Narrowing

106 Information area

107 Concave profile

108 Front of carrier bar

110 Automatic liquid changer

111 Carrier bar clamp

112 Opening and closing part

113 Pedestals

114 Fittings

115 vacancies

116 Receptacle

1 Positive pressure device

2 On-off valve

3, 4, 5, 6, 7, 8 Reservoir

9 Multi-channel switching valve

10 fluid exchange channels

11 Waste container

12 On-off valve

13 Negative pressure device

14 Waste channel

23, 24, 25, 26, 27, 28 Liquid supply channels.

Claims (15)

1. An automatic fluid changing device for supplying a fluid to and recovering a fluid from a carrier rod loaded with living cells before vitrification and/or during rewarming thawing, comprising:

the liquid storage containers are used for respectively storing various liquids required before vitrification freezing and/or in a rewarming and thawing process;

the multi-channel switching valve can selectively communicate each liquid storage container with a liquid exchange channel communicated to the carrying rod through a corresponding liquid supply channel or communicate the liquid exchange channel with a waste liquid channel used for discharging liquid out of the automatic liquid exchange device;

pumping means for providing a positive pressure for supplying liquid to the carrier bar and a negative pressure for recovering liquid from the carrier bar;

and the control module controls the multi-channel switching valve to alternately connect the liquid changing channel with each liquid supply channel, so that the liquid in the liquid storage container is supplied to the carrying rod under the action of the pumping device, and controls the multi-channel switching valve to communicate the liquid changing channel with the waste liquid channel after a corresponding preset time period so as to recover the liquid from the carrying rod under the action of the pumping device.

2. The automatic liquid changing device according to claim 1, wherein the liquid stored in the liquid storage container comprises all or a part of the following liquids: basic solution, frozen equilibrium solution, vitrified frozen solution, diluted solution, washing solution.

3. A self-changing fluid device according to claim 1, wherein said reservoir is replaceable or refillable.

4. The automatic liquid changing device according to claim 1, wherein the communication and disconnection of each liquid supply passage with the liquid changing passage are individually controlled by an on-off valve provided for each liquid supply passage.

5. The automatic liquid changing device according to claim 1, wherein the connection and disconnection of the waste liquid channel to and from the liquid changing channel are individually controlled by a switching valve provided for the waste liquid channel.

6. The automatic fluid changer of claim 1, wherein the multi-channel switching valve is a rotary valve.

7. The automatic liquid changing device according to any one of claims 1 to 6, wherein the pumping device comprises a positive pressure pump for providing positive pressure to the liquid supply channel when the liquid supply channel is communicated with the liquid changing channel, and a negative pressure pump for providing negative pressure to the waste channel when the waste channel is communicated with the liquid changing channel.

8. The automatic liquid changing device according to claim 7, wherein the pumping device provides a positive pressure continuously or only when each of the liquid supply channels is in communication with the liquid changing channel, and the pumping device provides a negative pressure continuously or only when the waste liquid channel is in communication with the liquid changing channel.

9. The automatic liquid changing device according to claim 2, wherein a switching valve associated with each of the liquid supply passages is disposed upstream or downstream of each of the liquid storage containers.

10. The automatic fluid exchange device of any one of claims 3 to 6, wherein each of the fluid reservoirs is integrated into a unitary replaceable reservoir assembly.

11. The automatic liquid changing device according to claim 10, further comprising a waste liquid container communicating with the multi-channel switching valve through the waste liquid channel for temporarily storing waste liquid from the waste liquid channel.

12. The automatic liquid changing device according to claim 11, wherein a switching valve associated with the waste liquid channel is disposed upstream or downstream of the waste liquid container.

13. The automatic liquid changing device of claim 11, wherein the waste liquid container is also integrated within the container assembly.

14. The automatic liquid changing device according to any one of claims 1 to 6, wherein the control module controls the solenoid valve associated with each liquid supply channel and the solenoid valve associated with the waste liquid channel so that each liquid supply channel and the waste liquid channel are kept disconnected when the multi-channel switching valve is switched.

15. The automatic liquid changing device according to claim 4 or 5, wherein the switching valve is a solenoid valve, a pneumatic valve or a hydraulic valve.

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