WO2014201694A1

WO2014201694A1 - Automatic operation method and system for rapid vitrification freezing of living cells

Info

Publication number: WO2014201694A1
Application number: PCT/CN2013/077674
Authority: WO
Inventors: 徐小杨
Original assignee: Xu Xiaoyang
Priority date: 2013-06-21
Filing date: 2013-06-21
Publication date: 2014-12-24
Also published as: CN104378979B; CN104378979A

Abstract

Disclosed is an automatic operation method for rapid vitrification freezing of living cells, comprising the following steps: (1) according to the information about the type of target cell to be frozen, obtaining a carrier corresponding to the type of target cell to be frozen, wherein the carrier is an ice carrier obtained through water coagulation/sublimation, and the carrier maintains a specific structure in the operating environment; (2) placing the liquid containing the target cell to be frozen onto the ice carrier to enable the fluid to be rapidly adsorbed by the ice carrier; and (3) until the thickness of the liquid containing the target cell to be frozen on the ice carrier reaches the set target thickness, transferring the ice carrier carrying the target to be frozen into a freezing medium so as to complete the rapid vitrification freezing. Also disclosed are an automatic operation device for rapid vitrification freezing of living cells and a carrier for rapid vitrification freezing of living cells.

Description

Automatic operation method and system for rapid vitrification of living cells

The invention relates to the field of vitrification and freezing technology, in particular to an automatic operation method and system for rapid vitrification of living cells, and a rapid vitrification carrier for living cells. Background technique

Rapid vitrification of living cells minimizes interference and damage to cellular biological properties. Taking the existing artificial reproductive technology as an example, the frozen fertilized egg or the embryo of the early division can be mechanically cooled, slowly cooled by a programmed cooling device, or fast vitrified freezing. In comparison, the programmed cooling technique is complicated, the equipment cost is high, the processing time is long, the amount of specimens processed is small, the vitrification is insufficient, and the damage to the cell microstructure is large, and the advantage is that the artificial dependence is small, and a small amount can be achieved. Parallel processing of specimens; rapid vitrification, rapid cooling rate, full vitrification, high recovery rate, but only manual operation, high skill requirements for operators, strict personnel skill training and high operational responsibility, easy Operational accidents have resulted in irregular embryo freezing and even embryo loss.

Take the manual rapid vitrification of embryos as an example. The specific method of the technology is divided into the following steps: Step 1: Cell dehydration, cryoprotectant balance treatment; Step 2: Prepare the pipette tool, cauterize the glass tube, manually stretch, prepare the cell pipette, the inner diameter is less than 1 mm; Step 3 : Prepare a liquid nitrogen container, fill in a sufficient amount of liquid nitrogen, adjust the microscope at the same time, and prepare the frozen carrier for use; Step 4: Manually aspirate the liquid containing the embryonic cell mass from the cryoprotectant using a cell pipette with a manual elastic tip. The amount of liquid required is as small as possible, about 10 to 30 microliters, to stably maintain the position of the liquid column in the cell pipette, the other hand stably holds the frozen carrier, and the liquid containing the embryonic cells is coated on the sheet carrier under a dissecting microscope. The target cells are identified and the excess liquid is aspirated, and the target cells are adhered to the carrier, and the carrier is quickly and manually inserted into the liquid nitrogen to complete the freezing and tube sealing. The main point of this method of operation is, first, the operating time is as short as possible. Living embryos are exposed to the external environment and have a direct effect on cell function. Second, try to smoke as little as possible. Take the liquid and absorb the excess liquid adsorbed on the carrier before the liquid nitrogen is introduced. The purpose is to make the liquid film around the cell as thin as possible, which is beneficial to increase the surface area directly exposed by the cells, thereby increasing the cooling rate. From the room temperature to the manual process of liquid nitrogen, the cooling rate is much higher than the cooling rate required for water crystallization in the cryoprotectant, and vitrification can be achieved. This method of operation relies on the skill of the operator, inefficiency, and the time during which the living embryo is directly exposed to the environment is related to the skill and proficiency of the operator. The operator has a high operational pressure when sucking excess liquid on the carrier. The stability requirements are high and the quality of the refrigeration is unpredictable. This operating system can only process several microliters of specimens at the same time, can not handle a large number of specimens, and severely limits the application range of vitrification technology.

Full and rapid completion of the vitrification process is a critical step in freezing living cells and retaining the physiological characteristics and functions of the cells. The existing vitrification technology has a small application range because of the limitation of the freezing carrier, resulting in low freezing efficiency, and only manual processing of small-capacity specimens can not be performed, and it cannot be used for simultaneous processing of large-capacity specimens. The commonly used vitrification technology requires osmotic dehydration treatment of cells, and is equilibrated with a cryoprotectant, and then enters rapid vitrification, but some cells are sensitive to the infiltration process, and osmotic dehydration directly destroys its functional structure, a more reasonable way. It is a direct freezing without cryoprotectant, such as direct vitrification of sperm cell suspension for rapid freezing.

The freezing efficiency is increased to be able to process hundreds of milliliters of specimens at a time. Vitrification technology can provide services in important areas such as long-term preservation of important stem cells, resuscitation or donation, and individualized blood component cells. Low-cost long-term stable storage, public-oriented solid hematopoietic stem cell banks, biological genetic information entity cell banks, long-term blood reservoirs built for disasters or wars, and more. The long-term retention of information on individual cell lines also provides an inexpensive and efficient physical reference frame for cell science and medical physiology research. Therefore, there is a need to develop an automated method and system for rapid vitrification of living cells.

SUMMARY OF THE INVENTION Embodiments of the present invention provide an automatic operation method and system for rapid vitrification of living cells, which can quickly and efficiently automate the rapid vitrification of living cells and improve the freezing efficiency. In order to achieve the above object, the embodiment of the present invention adopts the following technical solutions:

An automatic operation method for rapid vitrification of living cells, comprising the steps of:

51. Obtain a carrier corresponding to the type information of the frozen target cell according to the type information of the frozen target cell; wherein the carrier is an ice carrier formed by water coagulation/desublimation, and the carrier is in an operating environment Maintain a specific structure;

52. placing a liquid containing frozen target cells on the ice carrier, allowing the liquid to uniformly spread on the surface of the carrier, and being rapidly adsorbed by the ice carrier;

53. When the thickness of the liquid film containing the frozen target cells on the ice carrier reaches a set thickness, the ice carrier carrying the frozen target cells is transferred into the freezing medium to complete rapid vitrification. Correspondingly, an embodiment of the present invention further provides an automatic operating system for rapid vitrification of living cells, which is capable of performing the above operation method, including a central control device, a mechanical action device, and a freezing medium device and a carrier, wherein the carrier An ice carrier formed by coagulation/deposition of water, and the carrier maintains a specific structure under an operating environment; the central control device acquires a frozen target by reading a chip disposed outside a liquid container containing the frozen target cells The liquid information of the cell is determined according to the liquid information of the frozen target cell, and the carrier corresponding to the liquid information of the frozen target cell is determined, and a corresponding first control command is generated and sent to the mechanical action device; the mechanical action device is Receiving a control command to acquire a corresponding carrier, and taking a liquid containing the frozen target cells from the liquid container and placing the liquid on the ice carrier, so that the liquid is rapidly adsorbed by the ice carrier; until the inclusion on the ice carrier When the liquid film thickness of the frozen target cell reaches a set thickness, The central control device sends a second control command to the mechanical action device to cause the mechanical action device to move the ice carrier carrying the frozen target cells into the freezing medium device to complete rapid vitrification freezing.

In addition, an embodiment of the present invention further provides a living cell rapid vitrification freezing carrier, The carrier is an ice carrier formed by coagulation/decondensation of water, and the carrier maintains a sheet-like structure under an operating environment, and the carrier is used to carry the surface of the frozen target cells into a specific spatial microstructure.

An automatic operation method and system for rapid vitrification of living cells provided by an embodiment of the present invention, by using an ice carrier formed by water solidification/decondensation as a freezing carrier, can quickly adsorb due to the super-hydrophilic property of the ice carrier The liquid rapidly separates the frozen target cells from the liquid, thereby reducing the exposure time of the frozen target cells to the atmosphere, increasing the cooling rate, and reducing the freezing damage. In addition, due to the super-adsorption of the ice carrier, no need to manually absorb the excess liquid adsorbed on the carrier, so that the automatic operation of the rapid vitrification of the living cells can be realized, the working efficiency can be greatly improved, and the stability of the operation can be ensured. Timeliness and safety. BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings, which are incorporated in the claims Other drawings may also be obtained from these drawings without the use of creative labor.

1 is a flow chart showing an automatic operation method of rapid vitrification of living cells in Embodiment 1 of the present invention;

2 is a flow chart showing an automatic operation method of rapid vitrification of living cells in Embodiment 2 of the present invention;

3 is a structural block diagram of an automatic operating system for rapid vitrification of living cells in Embodiment 3 of the present invention;

Figure 4 is a schematic view showing the structure of a living cell fast vitrification and freezing carrier in the fourth embodiment of the present invention; Figure 5 is a schematic view showing the bearing surface structure of a living cell rapid vitrification freezing carrier shown in Figure 4;

Fig. 6 is a schematic view showing the structure of a living cell fast vitrification and freezing carrier in Example 5 of the present invention. Figure 7 is a schematic view showing the surface microstructure of the vitrified frozen carrier shown in Figure 6, which can be adjusted by micro The size and spatial shape of the column gaps are used to achieve different microvolume and surface characteristics. detailed description

BRIEF DESCRIPTION OF THE DRAWINGS The technical solutions in the embodiments of the present invention will be described in detail with reference to the accompanying drawings. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments of the present invention without creative work are within the scope of the present invention.

Example 1

Referring to FIG. 1, an embodiment of the present invention provides an automatic operation method for rapid vitrification of living cells. As shown in FIG. 1 , the method includes: Step S101: Acquire and freeze the target according to type information of frozen target cells. a carrier corresponding to the type information of the cell; wherein the carrier is an ice carrier formed by water coagulation/desublimation, and the carrier maintains a specific structure under an operating environment; and step S102, placing a liquid containing the frozen target cell On the ice carrier, the liquid is rapidly adsorbed by the ice carrier; step S103, until the liquid film thickness (capacity) of the frozen target cell on the surface of the ice carrier reaches a set thickness (capacity), The ice carrier carrying the frozen target cells is transferred into a freezing medium to complete rapid vitrification.

The present invention replaces the human work industry with the automatic operation method of the rapid vitrification of the living cells shown in Fig. 1, which can greatly improve the work efficiency and ensure the stability, timeliness and safety of the operation.

In order to provide a person skilled in the art to better understand the automatic operation method of the rapid vitrification of living cells of the present invention, the automatic operation method of the rapid vitrification of living cells of the present invention will be described in detail below with reference to FIG.

Example

2 is a flow chart of an automatic operation method for rapid vitrification of living cells in Embodiment 2 of the present invention, the method comprising: Step S201, obtaining a type information of the frozen target cell by reading a chip disposed outside a liquid container storing the frozen target cell;

Specifically, we know that the target cells need to be dehydrated before freezing, and the frozen target cells are placed in a container (injected with a cryoprotectant such as glycerin or DMSO) to balance the cryoprotectant with the frozen target. Free water in the cells to increase the freezing efficiency of the target cells and avoid crystal formation in the target cells.

Further, a chip is provided outside the liquid container containing the frozen target cells, and the chip pre-stores type information of the frozen target cells. Therefore, the type information of the frozen target cells can be obtained by reading a chip provided outside the liquid container in which the frozen target cells are stored.

Step S202, comparing the acquired type information of the frozen target cells with pre-stored information, thereby determining and acquiring a carrier corresponding to the type information of the frozen target cells; wherein the carrier is solidified/condensed by water An ice carrier, and the carrier maintains a specific structure in an operating environment;

Specifically, the type information of the different frozen target cells and the correspondence between the carriers may be preset, so that when the type information of the frozen target cells is acquired, the frozen target cells may be obtained by searching the corresponding relationship. The corresponding carrier type.

In this embodiment, the carrier is used to carry a frozen target cell for a freezing operation, which is an ice carrier solidified/condensed by purified water, and the carrier maintains a specific structure in an operating environment. The ice carrier solidified/condensed from pure water is super-hydrophilic and can quickly adsorb liquid. The ice carrier is exposed for a short period of time under operating conditions (e.g., -2 degrees Celsius) sufficient to maintain a substantially fixed sheet structure. In addition, the ice carrier is used to carry a surface of the frozen target cell with a specific spatial structure (for example, a porous or surface microscopic structure) to increase the surface area of the carrier adsorbed liquid and improve the adsorption performance. The ice carrier includes a carrier portion and a restriction portion projecting from the carrier portion, the restriction portion for limiting a thickness of a liquid containing the frozen target cells placed on the carrier portion. The specific structure of the carrier will be described in detail later with reference to Figs. Step S203, placing a liquid containing frozen target cells on the ice carrier, so that the liquid is rapidly adsorbed by the ice carrier; Specifically, an appropriate amount of liquid (including frozen target cells) is taken out from the container containing the frozen target cell liquid, and then placed on the corresponding carrier. Since the carrier is a super-hydrophilic ice carrier, when a liquid containing frozen target cells is placed on the ice carrier, the liquid is rapidly adsorbed by the ice carrier, thereby realizing the same with the frozen target cell. Rapid separation to expose the frozen target cells. Step S204, when the liquid film thickness (capacity) of the frozen target cell on the surface of the ice carrier reaches a set thickness (capacity), the ice carrier carrying the frozen target cell is moved into the freezing medium to complete rapid vitrification. Freezing; Specifically, in this step, the following two situations may be included:

When the frozen target cell is a cell or a collection of cells that need to expose the largest surface area, immediately after the frozen target cell is sufficiently exposed, the ice carrier carrying the frozen target cell is immediately transferred into a freezing medium (for example, liquid nitrogen). Rapid vitrification; or

When the frozen target cell is a cell or a collection of cells that is suitable for simultaneous freezing with an environmental liquid, when the liquid volume of the frozen target cell to be contained is less than or equal to the capacity determined by the restriction portion, the frozen target cell is immediately carried. The ice carrier is transferred into a freezing medium (for example, liquid nitrogen) to complete rapid vitrification.

Step S205, performing vacuum freeze-drying treatment on the frozen target cells that have been subjected to rapid vitrification to sublimate the ice carrier carrying the frozen target cells into water vapor, thereby realizing the lyophilized powder state of the frozen target cells. save.

In the frozen sperm suspension, blood component cell collection, etc., a subsequent freeze-drying process is required. For human assisted reproductive technology, the quality of embryo cryopreservation can be improved. After sperm freezing, it can be directly lyophilized under low temperature, and sperm cells can be stored for a long time at a lower cost of lyophilized powder. For the preservation of blood components. The specimens can be processed in hundreds of milliliters simultaneously. After the treated specimens are sublimed and vacuumed, the blood components can be stably stored for a long time, and the storage cost and the reprocessing cost are greatly reduced.

Since vacuum freeze-drying is familiar to those skilled in the art, it will not be described in detail herein.

So far, the rapid vitrification process of living cells has been completed. Thereafter, the frozen target cells can be resuscitated using a defrosting protectant. 2 is a flow chart showing an automatic operation method for rapid vitrification of living cells according to an embodiment of the present invention. It should be understood that the steps of the automatic operation method for rapid vitrification of living cells proposed by the present invention are not limited to FIG. 2 . In the order of execution shown, those skilled in the art can arbitrarily change the automated method steps of rapid vitrification of living cells in accordance with the teachings of the present invention.

The above is a preferred embodiment of the present invention, and it should be noted that those skilled in the art can also make several improvements and retouchings without departing from the principles of the present invention. These improvements and retouchings are also considered. It is the scope of protection of the present invention.

Example 3

Referring to FIG. 3, a structural block diagram of an automatic operating system for rapid vitrification of living cells in Embodiment 3 of the present invention. It can be understood that the automatic operating system of the embodiment can execute the above automatic operation method. As shown in FIG. 3, the automatic operating system 10 includes a central control device 1, a mechanical action device 2, and a freezing medium device 3 and a carrier 4, wherein the carrier 4 is used to carry frozen target cells for performing a freezing operation, which is An ice carrier formed by solidification/condensation of purified water, and the carrier maintains a specific structure in an operating environment. The ice carrier solidified/condensed from pure water is super-hydrophilic and can quickly adsorb liquid. The ice carrier is maintained in a fixed sheet-like structure under an operating environment (preferably 0 degrees Celsius) to prevent the ice carrier from being melted and deformed. In addition, the ice carrier is used to carry the surface of the frozen target cell as a porous structure or other suitable spatial structure, such as a surface microscopic structure having a specific capacity, to increase the surface area of the adsorbed liquid of the carrier, and to improve adsorption. performance. The ice carrier includes a carrier portion and a restriction portion projecting from the carrier portion, the restriction portion for limiting a thickness of the vitrified liquid containing the frozen target cells placed on the carrier portion. The specific structure of the carrier will be described in detail later with reference to Figs. In the embodiment, the central control device 1 acquires the vitrified liquid information of the frozen target cells by reading a chip disposed outside the vitrified liquid container containing the frozen target cells, according to the frozen target cells. The vitrification liquid information determines a carrier corresponding to the vitrified liquid information of the frozen target cell, and generates a corresponding first control command to be transmitted to the mechanical action device 2. In a specific implementation, the central control device 1 is provided with a database, and the database stores different freeze target details. Corresponding relationship between the vitrification liquid information of the cell and the carrier; the central control device 1 searches for the correspondence in the data according to the obtained vitrification liquid information of the frozen target cell, thereby judging the cell with the frozen target The vitrified liquid information corresponds to the type of carrier 4. The mechanical action device 2 acquires a corresponding carrier 4 according to the received first control instruction, and takes out a vitrification liquid containing frozen target cells from the vitrification liquid container, and places the same on the ice carrier. The vitrified liquid is rapidly adsorbed by the ice carrier.

Specifically, the mechanical action device 2 obtains a corresponding frozen carrier according to the received first control command, and then takes out an appropriate amount of liquid (including frozen target cells) from the vitrified liquid container containing the frozen target cells. Placed on the corresponding carrier. Since the carrier is a super-hydrophilic ice carrier, when a vitrification liquid containing frozen target cells is placed on the ice carrier, the vitrification liquid is rapidly adsorbed by the ice carrier, thereby achieving Rapid separation of frozen target cells to expose the frozen target cells. The central control device 1 sends a second control command to the mechanical action device 2 until the liquid film thickness (capacity) containing the frozen target cells on the surface of the ice carrier reaches a set thickness (capacity). The mechanical action device 2 moves the ice carrier carrying the frozen target cells into the freezing medium device 3 (for example, a liquid nitrogen device) to complete rapid vitrification.

Specifically, when the frozen target cell is a cell or a cell aggregate that needs to expose a maximum surface area, after the frozen target cell is sufficiently exposed, the mechanical action device 2 immediately carries the second control instruction according to the received second control instruction. Freezing the target carrier's ice carrier into a freezing medium (eg, liquid nitrogen) to complete rapid vitrification; or

When the frozen target cell is a cell or a collection of cells that are simultaneously frozen with the vitrifying liquid, the mechanical action of the vitrified liquid to be included in the frozen target cell is less than or equal to the capacity determined by the restriction portion The device 2 immediately moves the ice carrier carrying the frozen target cells into a freezing medium (for example, liquid nitrogen) according to the received second control command to complete rapid vitrification freezing.

Preferably, in the embodiment, the automatic operating system further comprises a vacuum freeze-drying device 5, after the ice carrier of the frozen target cell is moved into the freezing medium device 3 to complete rapid vitrification, The central control device 1 sends a third control command to the mechanical action device 2, causing the mechanical action device 2 to move the frozen target cells (along with the carrier) into the vacuum freeze-drying device 5 for vacuum freeze-drying The drying treatment is performed to sublimate the ice carrier carrying the frozen target cells into water vapor, thereby achieving lyophilized powder state preservation of the frozen target cells.

Specifically, in the frozen sperm suspension, blood component cell collection, etc., a vacuum freeze-drying device 5 is required to perform a subsequent freeze-drying process. For human assisted reproductive technology, the quality of embryo cryopreservation can be improved. After sperm freezing, it can be directly lyophilized under low temperature, and sperm cells can be stored for a long time at a lower cost of lyophilized powder. For the preservation of blood components. The specimens in hundreds of milliliters can be processed simultaneously. After the treated specimens are sublimed and vacuumed, the blood components can be stably stored for a long time, and the storage cost and the reprocessing cost are effectively reduced.

The automatic operating system for rapid vitrification of living cells of the present embodiment can automatically complete the entire freezing operation process of the frozen target cells by reading the information in the chip provided outside the vitrified liquid container containing the frozen target cells, without Manual participation greatly improves work efficiency and ensures operational stability, timeliness and safety.

Example 4

Referring to Figures 4 to 5, a schematic diagram of the structure of a living cell rapid vitrification carrier in Embodiment 4 of the present invention is shown. As shown in Fig. 4, the carrier 4 of the present embodiment can be used in the above-described automatic operation method and system for rapid vitrification of living cells to carry frozen target cells for a fully automatic freezing operation. The carrier is an ice carrier solidified/condensed from purified water, and the carrier maintains a specific structure in an operating environment. The ice carrier solidified/condensed from pure water is super-hydrophilic and can quickly adsorb liquid. The ice carrier is maintained in a fixed sheet-like structure under an operating environment (e.g., -2 degrees Celsius) to prevent the ice carrier from being melted and deformed.

Specifically, the carrier 4 includes a carrying portion 41 and a restricting portion 42 protruding from the carrying portion 41, and the carrying portion 41 is configured to carry a surface of the frozen target cell as a porous structure 411 (refer to FIG. 5), Increasing the surface area of the carrier adsorbed liquid and improving the adsorption performance. The restricting portion 42 is a plurality of convex protrusions on the carrying portion 41 for limiting the glass containing the frozen target cells placed on the carrying portion 41 Liquid thickness.

The carrier 4 provided in this embodiment has super hydrophilic surface characteristics. The hydrophilic material is first perforated, and the size of the pore size is determined by the characteristics of the frozen specimen, so that the aqueous solution can be quickly adsorbed and uniformly distributed into a thin film, thereby rapidly and automatically exposing the target cells, and maximizing the exposed surface area of the target cells. For target cells that need to be frozen simultaneously with the solution, the above structure can enhance the adhesion of the liquid on the carrier, increase the flatness and adhesion of the liquid film, and withstand the acceleration of the mechanical system action, ensuring that the specimen (freezing target cells) is not lost. , to ensure the quality of the frozen.

The method of using the vector of the present embodiment is as follows: For freezing of a target cell or a collection of cells that need to expose a maximum surface area, the liquid containing the target cell or cell collection is directly transferred to the functional surface of the carrier, after the target cell is sufficiently exposed, Immediately transferred to a frozen working environment (eg, liquid nitrogen environment); for a cell or collection of cells that are suitable for simultaneous freezing with the culture fluid environment liquid, the liquid volume containing the target cell or collection of cells is less than or equal to the liquid film thickness restriction portion 42 When the carrier capacity is automatically determined, the carrier carrying the target specimen (frozen target cells) is immediately transferred to the frozen working environment to complete the freezing.

Example 5

Referring to Figures 6-7, there is shown a schematic view of the structure of a living cell fast vitrification carrier in Example 5 of the present invention. As shown in Fig. 6, the carrier 4 of the present embodiment can be used in the above-described automatic operation method and system for rapid vitrification of living cells to carry frozen target cells for a fully automatic freezing operation. The carrier is made of a hydrophilic polymer film (e.g., a polycarbonate film material) and is a two-layer composite having a specific surface structure with a layer spacing capable of rapidly adsorbing a liquid (Fig. 7).

This embodiment differs from the embodiment 4 in that the carrier 4 is composed of a plurality of carriers of the embodiment 4, and therefore, the carrier of the embodiment is suitable for carrying a large cell or a plurality of target cells for freezing.

As can be seen from Example 4 and Example 5, the carrier of the present invention is composed of different types of carriers by its size, the thickness of the liquid film which is restricted by the restriction portion, and the pore size of the bearing surface, and is thus suitable for different frozen target cells. In summary, the automatic operation of rapid vitrification of living cells provided by the embodiments of the present invention is provided. The method and system adopt the ice carrier formed by water solidification/desublimation as a freezing carrier, and the super-hydrophilic property of the ice carrier can rapidly adsorb the liquid, so that the frozen target cells and the liquid are quickly separated, thereby reducing the exposure of the frozen target cells. In the atmospheric environment, increase the rate of cooling and reduce freezing damage. In addition, due to the super-adsorption of the ice carrier, no need to manually absorb the excess liquid adsorbed on the carrier, so that the automatic operation of the rapid vitrification of the living cells can be realized, the working efficiency can be greatly improved, and the stability of the operation can be ensured. Timeliness and safety.

The invention has been described above in connection with the preferred embodiments, but the invention is not limited to the embodiments disclosed above, but should be construed to cover various modifications and equivalent combinations in accordance with the nature of the invention.

Claims

1. An automatic method for rapid vitrification of living cells, comprising the steps of:

52. placing a liquid containing frozen target cells on the ice carrier, allowing the liquid to be rapidly adsorbed by the ice carrier;

53. When the thickness of the liquid film containing the frozen target cells on the surface of the ice carrier reaches a set thickness, the ice carrier carrying the frozen target cells is transferred into a freezing medium to complete rapid vitrification.

2. The automatic operation method for rapid vitrification of living cells according to claim 1, wherein after the step S3, the method further comprises the steps of:

54. Performing a vacuum freeze-drying process on the frozen target cells that have completed rapid vitrification, together with a carrier, to sublimate the ice carrier carrying the frozen target cells into water vapor, thereby realizing the lyophilized powder of the frozen target cells. The status is saved.

The automatic operation method of the rapid vitrification of the living cells according to claim 1, wherein the step S1 comprises the following steps:

511. Obtaining type information of the frozen target cell by reading a chip or a barcode disposed outside a liquid container storing the frozen target cell type;

512. Compare the acquired type information of the frozen target cells with pre-stored information, thereby determining and acquiring a carrier and cryoprotectant treatment procedure corresponding to the type information of the frozen target cells.

4. The automatic method for rapid vitrification of living cells as claimed in claim 1, wherein The ice carrier is a sheet-like structure, and the surface of the ice carrier for carrying the frozen target cells is a porous structure or a surface microscopic structure having a specific capacity.

5. The automatic operation method for rapid vitrification of living cells according to claim 4, wherein the ice carrier comprises a carrying portion and a restricting portion protruding from the carrying portion, wherein the restricting portion is Limiting the thickness of the liquid film comprising frozen target cells placed on the surface of the carrier.

6. The method of claim 3, wherein the step S3 specifically comprises:

531. When the frozen target cell is a cell or a cell aggregate that needs to expose a maximum surface area, after the frozen target cell is sufficiently exposed, the vector carrying the frozen target cell is immediately transferred into a freezing medium to complete rapid vitrification; Or

532. When the frozen target cell is a cell or a cell aggregate that is suitable for freezing simultaneously with a cell environment liquid, when the liquid volume of the vitrified liquid to be frozen target cells is less than or equal to the capacity determined by the restriction portion, immediately The ice carrier carrying the frozen target cells is transferred into a freezing medium to complete rapid vitrification.

7. An automated method of rapid vitrification of living cells as claimed in claim 1, wherein the temperature of the operating environment is maintained below the natural freezing point temperature of the local purified water.

8. An automated method of rapid vitrification of living cells as claimed in claim 1, wherein said freezing medium is liquid nitrogen.

9. An automatic operating system for rapid vitrification of living cells, comprising: a central control device, a mechanical action device, and a freezing medium device and a carrier, wherein the carrier is ice formed by water solidification/condensation a carrier, and the carrier maintains a specific structure in an operating environment; The central control device acquires type information of the frozen target cells by reading a chip or a barcode disposed outside the container in which the frozen target cells are stored, and determines the type information of the frozen target cells according to the type information of the frozen target cells. Corresponding carrier, and generating a corresponding first control command to be sent to the mechanical action device; the mechanical action device acquiring a corresponding carrier according to the received first control command, and taking out the frozen from the vitrified liquid container a liquid of the target cell is placed on the ice carrier, and the vitrification liquid is rapidly adsorbed by the ice carrier; until the thickness of the liquid film containing the frozen target cell on the surface of the ice carrier reaches a set thickness The central control device sends a second control command to the mechanical action device to cause the mechanical action device to move the ice carrier carrying the frozen target cells into the freezing medium device to complete rapid vitrification.

10. The automatic operating system for rapid vitrification of living cells according to claim 9, further comprising a vacuum freeze-drying device, wherein the ice carrier of the frozen target cell is moved into the freezing medium device to complete the fast After vitrification, the central control device sends a third control command to the mechanical action device, and the mechanical action device moves the frozen target cells together with the carrier into the vacuum freeze-drying device for vacuum freeze-drying Processing to sublimate the ice carrier carrying the frozen target cells into water vapor, thereby achieving lyophilized powder state preservation of the frozen target cells.

11. The automatic operating system for rapid vitrification of living cells according to claim 9, wherein the central control device is provided with a database, and the database stores different types of information and carriers of the frozen target cells. Correspondence between the two; the central control device performs a search in the data based on the acquired type information of the frozen target cells, thereby determining a carrier corresponding to the type information of the frozen target cells.

12. The automatic operating system for rapid vitrification of living cells according to claim 9, wherein The ice carrier is a sheet-like structure, and the surface of the ice carrier for carrying the frozen target cells is a porous structure or a surface fine three-dimensional structure having a specific volume.

13. The automatic operating system for rapid vitrification of living cells according to claim 12, wherein the ice carrier comprises a carrying portion and a restricting portion protruding from the carrying portion, the restricting portion being used for A liquid film thickness comprising frozen target cells placed on the surface of the carrier is limited.

The automatic operating system for rapid vitrification of living cells according to claim 13, wherein the thickness of the liquid film containing the frozen target cells on the surface of the ice carrier reaches a set thickness :

When the frozen target cell is a cell or a collection of cells that need to expose a maximum surface area, after the frozen target cell is sufficiently exposed; or

When the frozen target cell is a cell or a collection of cells suitable for simultaneous freezing with an environmental liquid, the liquid volume of the environmental liquid to be frozen-containing the target cell is less than or equal to the capacity determined by the restriction.

15. The automated operating system for rapid vitrification of living cells according to claim 9, further comprising operating environment control means for maintaining the temperature of the operating environment below the natural freezing temperature of the local purified water.

The automatic operating system for rapid vitrification of living cells according to claim 9, wherein the freezing medium is liquid nitrogen.

17. A living cell rapid vitrification freezing carrier, characterized in that the carrier is an ice carrier formed by water coagulation/desublimation, and the carrier maintains a sheet-like structure in an operating environment, and the carrier is used The surface carrying the frozen target cell is a porous structure or other specific spatial structure to increase water The rate at which the solution diffuses at the surface or has a specific capacity.

18. The living cell rapid vitrification freezing carrier according to claim 17, wherein the ice carrier comprises a bearing portion and a restricting portion protruding from the bearing portion, the restricting portion for restricting placement The thickness of the liquid film on the surface of the carrier portion containing the frozen target cells.

The living cell rapid vitrification carrier according to claim 18, wherein the restriction portion is plural.

20. The living cell fast vitrification freezing carrier of claim 17, wherein the temperature of the operating environment is maintained below a temperature at which the local purified water naturally freezes.