CN216722872U

CN216722872U - Vitrification freezing carrier

Info

Publication number: CN216722872U
Application number: CN202121456505.0U
Authority: CN
Inventors: 杨云; 朱波风; 李维杰
Original assignee: Zhejiang Mingyue Medical Technology Co ltd; Shanghai Mingyue Medical Technology Co ltd
Current assignee: Zhejiang Mingyue Medical Technology Co ltd; Shanghai Mingyue Medical Technology Co ltd
Priority date: 2021-06-29
Filing date: 2021-06-29
Publication date: 2022-06-14
Anticipated expiration: 2031-06-29

Abstract

The utility model relates to a vitrification freezing carrier, which comprises a carrying rod, a carrying piece and a sealing piece, wherein the carrying piece is arranged at one end of the carrying rod and is provided with a groove; the sealing piece is used for being attached to the slide glass so as to seal the groove. According to the vitrification freezing carrier, the groove of the slide glass is used for placing the oocyte or embryo sample, the phenomenon that the slide glass shakes to drop eggs or embryos can be avoided, and when the redundant liquid of the sample is removed, the oocyte or embryo sample in the groove cannot shift, so that the operation process is simpler, more convenient and more stable, and the efficiency of transferring the oocyte or embryo sample is higher. And after the sample is transferred to the groove and before the sample is transferred to liquid nitrogen, the sealing sheet is attached to the slide glass to seal the groove, so that oocytes and/or embryos in the sample are not directly contacted with the liquid nitrogen in the vitrification cryopreservation process, the problem that cross contamination of other samples is caused by potential pollutants through the liquid nitrogen is avoided, and the vitrification freezing reliability is improved.

Description

Vitrification freezing carrier

Technical Field

The utility model relates to the technical field of medical instruments, in particular to a vitrification freezing carrier.

Background

In recent years, along with the reasons of environmental pollution, late childbearing age, life pressure and the like, infertility becomes a common disease of the breeding age couples in modern society. The human assisted reproduction technology, which is the most representative technology of reproduction medicine, has become an effective, even nearly irreplaceable clinical means for treating infertility through the development of many years. Cryopreservation of oocytes or embryos is a method to preserve female fertility and is also an important link in assisted reproductive technology.

At present, vitrification is the first choice method for preserving oocytes or embryos in a freezing way in clinic, on one hand, vitrification is the process of directly transforming a sample from a liquid state to a glass state by using high-concentration vitrification refrigerating fluid at a very fast cooling rate, and the process reduces the ice crystal damage to cells to the maximum extent; on the other hand, compared with programmed freezing, the vitrification freezing efficiency is high and the cost is low. The traditional vitrification freezing carrier is over simple in design, and has the problems of inconvenient operation, cross contamination of a plurality of samples stored in liquid nitrogen and the like.

SUMMERY OF THE UTILITY MODEL

Accordingly, there is a need for a vitrified frozen carrier and a method of cryopreservation that are easy to handle and can avoid the problem of cross contamination.

A vitrified frozen carrier comprising:

a carrier bar;

the slide glass is arranged at one end of the slide rod and is provided with a groove; and

and the sealing piece is attached to the slide glass to seal the groove.

In some of these embodiments, the carrier sheet includes a sheet body region and a recess region, the recess being provided in the recess region;

the thickness of the carrier sheet in the groove area is smaller than that in the sheet material body area.

In some of these embodiments, the slide has a thickness in the region of the recess of 50 μm to 200 μm; the thickness of the carrier sheet in the area of the sheet body is 200-500 mu m.

In some of these embodiments, the groove has a radial slot dimension of 100 μm to 200 μm; and/or

The depth of the groove is 150-200 μm.

In some of these embodiments, the grooves are cylindrical or prismatic; and/or

The bottom wall of the groove is a plane or a concave cambered surface.

In some of these embodiments, the recess on the slide is multiple, and the sealing sheet is used to seal each recess on the slide.

In some of these embodiments, the sealing panel is a transparent sealing panel; and/or

The thickness of the sealing piece is 50-100 mu m.

In some embodiments, the carrier rod comprises a holding section and a marking section connected with the holding section, the end of the carrier rod where the holding section is located is connected with the slide, and the marking section is used for marking sample information.

In some embodiments, the mark segment is provided with an information identification electronic tag for storing sample information.

In some of these embodiments, the information identifying electronic label comprises at least one of a bar code, a two-dimensional code, and an electronic chip label.

In some of these embodiments, the length of the gripping section of the carrier bar is adjustable.

In some of these embodiments, the gripping section of the carrier bar is provided with a non-slip texture.

In some embodiments, the material of the slide is at least one of polypropylene, polystyrene, polyethylene terephthalate, polyethersulfone resin, styrene thermoplastic elastomer, styrene, and polyethylene terephthalate-1, 4-cyclohexanedimethanol.

A vitrification cryopreservation method adopting the vitrification freezing carrier comprises the following steps:

transferring the sample treated by the vitrified refrigerating fluid into the groove of the slide glass, and absorbing redundant liquid in the groove;

placing the sealing sheet on a slide sheet with a configured sample, and attaching the sealing sheet to the slide sheet to seal the groove;

and (4) holding the carrying rod, and transferring the vitrified frozen carrier with the configured sample into liquid nitrogen for preservation.

In some of these embodiments, the vitrified chilled liquid comprises a first chilled liquid and a second chilled liquid;

the first refrigerating fluid comprises a base fluid and a permeability protective agent, and the second refrigerating fluid comprises a base fluid, a permeability protective agent and a non-permeability protective agent;

wherein the osmotic protective agent comprises ethylene glycol and dimethyl sulfoxide, and the non-osmotic protective agent comprises sucrose; the base solution comprises TCM199, 10 mM-20 mM HEPES and 0.1% -0.5% (w/w) human serum albumin, or DMEM, 10 mM-20 mM HEPES and 0.1% -0.5% (w/w) human serum albumin.

In some of these embodiments, the first freezing fluid comprises 5% to 8% (v/v) ethylene glycol, 5% to 8% (v/v) dimethyl sulfoxide, and 84% to 90% (v/v) base fluid;

the second refrigerating fluid comprises 12-20% (v/v) glycol, 12-20% (v/v) dimethyl sulfoxide, 0.5-1.5M cane sugar and 60-76% (v/v) base fluid.

In some embodiments, the sample is sequentially processed by the first freezing liquid and the second freezing liquid, and the equilibration time of the sample in the first freezing liquid and the equilibration time of the sample in the second freezing liquid are respectively 3min to 5min and 30s to 60 s.

The slide glass of the vitrification freezing carrier provided by the utility model is provided with the groove for placing the oocyte or embryo sample, so that the phenomenon of egg dropping or embryo dropping caused by the shaking of the slide glass can be avoided, and meanwhile, when the redundant liquid of the sample is removed, the oocyte or embryo sample in the groove can not be displaced, so that the operation process is simpler, more convenient and more stable, and the efficiency of transferring the oocyte or embryo sample is higher. Simultaneously, after the sample shifts to in the recess and before shifting to in the liquid nitrogen, adopt the sample of gasket and slide glass laminating in order to seal the recess, and then make oocyte and/or embryo in the sample freeze the in-process and directly not contact with the liquid nitrogen vitrifyingly, avoid potential pollutant to lead to the fact the cross contamination's of other samples problem through the liquid nitrogen, so effectively solved the cross contamination problem, improved the refrigerated reliability of vitrification.

Drawings

FIG. 1 is a schematic diagram of the structure of a vitrified frozen carrier according to an embodiment of the utility model;

FIG. 2 is a schematic structural view of a slide of the vitrified frozen carrier shown in FIG. 1;

FIG. 3 is a schematic diagram of the structure of the slide and sealing plate of the vitrified frozen carrier shown in FIG. 1;

FIG. 4 is a schematic cross-sectional view of the chip shown in FIG. 2;

FIG. 5 is a schematic cross-sectional structure of a carrier sheet according to another embodiment of the present invention;

FIG. 6 is a schematic structural diagram of a carrier bar according to an embodiment of the present invention;

FIG. 7 is a schematic view of the structure of a carrier rod of the vitrified frozen carrier shown in FIG. 1;

FIG. 8 is a schematic structural view of a carrier bar according to yet another embodiment of the present invention;

FIG. 9 is a schematic structural view of a control product;

figure 10 is a graph of the cooling rate results for the frozen carriers of the utility model and the control product during use.

Detailed Description

To facilitate an understanding of the utility model, the utility model will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

In the description of the present invention, it is to be understood that the terms "length," "thickness," "upper," "lower," "top," "bottom," "inner," and the like are used in the orientations and positional relationships indicated in the drawings for convenience in describing the present invention and simplicity in description, and do not indicate or imply that the referenced devices or elements must have a particular orientation, be constructed in a particular orientation, and be operated, and thus are not to be construed as limiting the present invention.

Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.

In the present invention, unless otherwise expressly stated or limited, the terms "connected," "secured," and the like are to be construed broadly and can, for example, be fixedly connected, detachably connected, or integral to one another; they may be mechanically coupled, directly coupled, indirectly coupled through intervening media, coupled between two elements, or coupled in any other manner that does not materially affect the operation of the device, unless otherwise specifically limited. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations. In the present invention, unless otherwise expressly stated or limited, the first feature "on" or "under" the second feature may be directly contacting the first and second features or indirectly contacting the first and second features through an intermediate.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the utility model herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the utility model. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Referring to fig. 1, one embodiment of the present invention provides a vitrified frozen carrier 10, which comprises a slide 100, a carrying rod 200 and a sealing plate 300 (shown in fig. 3).

The chip 100 is disposed at one end of the rod 200. Referring to fig. 2, a slide 100 has a groove 101.

Referring to fig. 3, the sealing plate 300 is used to attach to the slide 100 to seal the groove 101.

The slide 100 of the conventional vitrified frozen carrier 10 is generally a flat sheet or a sheet with a small curvature, and the transfer operation of the sample such as the oocyte or the embryo is very difficult, and the egg drop or the embryo drop is very easy to occur. The conventional technique is to directly load the sample such as oocyte or embryo processed by vitrified refrigerating fluid on the slide 100, suck the redundant fluid of the sample, and then insert the sample into liquid nitrogen for storage. This method presents the risk of cross-contamination of multiple samples during vitrification cryopreservation, since the samples are in direct contact with liquid nitrogen during storage.

Based on this, the slide 100 of the vitrification freezing carrier 10 provided by the utility model is provided with the groove 101 for placing the oocyte or embryo sample, so that the phenomenon of egg dropping or embryo dropping caused by shaking of the slide 100 can be avoided, and meanwhile, when the redundant liquid of the sample is removed, the oocyte or embryo sample in the groove 101 cannot be displaced, so that the operation process is simpler and more stable, and the efficiency of transferring the oocyte or embryo sample is higher. Simultaneously, after the sample shifts to in recess 101 and before shifting to in the liquid nitrogen, adopt the sample in gasket 300 and slide glass 100 laminating in order to sealed recess 101, and then make oocyte and/or embryo in the sample freeze the in-process and directly not contact with the liquid nitrogen vitrifyingly, avoid potential pollutant to lead to the fact the cross contamination's of other samples problem through the liquid nitrogen, so effectively solved the cross contamination problem, improved the refrigerated reliability of vitrification.

With continued reference to fig. 2, in some embodiments, the carrier 100 includes a sheet body region 110 and a recessed region 120, and the recess 101 is disposed in the recessed region 120. Referring to fig. 4 and 5, the thickness of the carrier sheet 100 in the recessed area 120 is smaller than the thickness of the carrier sheet 100 in the sheet body area 110, i.e., the thickness between the inner bottom surface of the recess 101 of the carrier sheet 100 (i.e., the bottom wall of the recess 101) and the surface opposite to the inner bottom surface (i.e., the outer bottom surface) is smaller than the thickness of the carrier sheet 100 in the sheet body area 110. Thus, the thicker sheet body region 110 of the slide 100 provides sufficient bearing capacity, the thickness of the groove region 120 is reduced, the temperature reduction rate is greatly improved as much as possible by the locally thinner structure of the groove 101, and the sample in the sealed groove 101 achieves a better low-temperature preservation effect without affecting the bearing capacity of the whole slide 100.

It is understood that the carrier bar 200 is used to hold the indicia of manipulation and sample information; slide 100 is used to carry a sample such as an oocyte and/or embryo. The slide bar 200 and the slide 100 may be integrally or detachably connected. In view of simplifying the manufacturing process and reducing the manufacturing cost, a detachable structure, such as a snap or screw connection, is preferable to prevent the slide bar 200 and the slide 100 from falling off, sliding, and the like during the operation or storage.

Further, the sealing sheet 300 and the slide sheet 100 may be bonded to each other by heat pressing, adhesion, or the like.

In some of these embodiments, the slide 100 has a thickness in the recessed region 120 of 50 μm to 200 μm; the carrier sheet 100 has a thickness of 200 to 500 μm in the sheet body region 110.

Further, the thickness of the chip 100 in the groove area 120 is 80 μm to 100 μm. Further, the carrier sheet 100 has a thickness of 250 to 400 μm in the sheet body region 110.

The material of the slide 100 is preferably food grade or medical grade high impact polymer material in consideration of the bearing capacity, biocompatibility and high cooling rate required by the slide 100. Further, the material of the slide sheet 100 is preferably transparent.

Further, the material of the carrier sheet 100 is at least one of PP (polypropylene), PS (polystyrene), PET (polyethylene terephthalate), PES (polyethersulfone resin), SBS (styrene-based thermoplastic elastomer), SBC (styrene), and PCTG (polyethylene terephthalate-1, 4-cyclohexanedimethanol ester).

Further, the material of the carrier sheet 100 in the sheet body region 110 and the recessed region 120 may be the same or different.

In some of these embodiments, the radial dimension of the notches of the grooves 101 is between 100 μm and 200 μm. In some of the embodiments, the groove 101 is cylindrical or prismatic, and the sidewall shape of the groove 101 is a cylindrical side or a prismatic side, but is not limited thereto. For example, the radial dimension of the groove 101 for a cylindrical notch is the circular diameter of the notch. In some of these embodiments, the bottom wall of the groove 101 is flat (as shown in fig. 4) or concavely curved (as shown in fig. 5).

Further, the outer bottom surface of the bottom wall of the recess 101 is flush with the surface of the sheet body region 110, which minimizes the thickness of the carrier sheet 100 in the recess 101 while maintaining the carrying capacity. In particular, the inner bottom surface of the groove 101 of the carrier 100 and the other surface (i.e., the outer bottom surface) opposite to the inner bottom surface are both flat surfaces, so as to facilitate the processing, production and processing, that is, the carrier of the present application can be implemented by directly forming the groove 101 on flat PP (polypropylene), PS (polystyrene), PET (polyethylene terephthalate), PES (polyethersulfone resin), SBS (styrene thermoplastic elastomer), SBC (styrene), and PCTG (polyethylene terephthalate-1, 4-cyclohexanedimethanol).

In some of these embodiments, the grooves 101 have a depth of 150 μm to 200 μm, and in these embodiments, the depth of the grooves 101 may be greater than the thickness of the sheet body region 110, such as by deep drawing the slide 100 or by forming each groove 101 in a molten state into a groove region of the lower ballast sheet 100. The stability of the operation process can be further improved by the preference for the radial dimension and/or depth of the groove 101.

In some of these embodiments, the recesses 101 on the slide 100 are multiple and the sealing plate 300 is used to seal each recess 101 on the slide 100 at the same time. The sealing of each groove 101 is realized by one sealing sheet 300, and the sealing is simple and convenient.

Further, a plurality of grooves 101 are distributed in an array on the chip 100. Specifically, the plurality of grooves 101 are arranged in a plurality of rows and a plurality of columns in two directions perpendicular to each other.

In some of these embodiments, sealing panel 300 is a transparent sealing panel 300. The transparent sealing sheet 300 is easy to observe.

In some of these embodiments, the sealing disc 300 has a thickness of 50 μm to 100 μm.

In some embodiments, the sealing plate 300 can be made of the same material as the slide 100.

With continued reference to fig. 1, in some embodiments, the slide bar 200 includes a holding section 210 and a marking section 220 connected to the holding section 210, the slide bar 200 is connected to the slide 100 at an end where the holding section 210 is located, and the marking section 220 is used for marking the sample information.

The holding section 210 and the marking section 220 may be integrally formed, or detachably formed, such as by a snap or screw connection, or may be connected in other manners.

It is understood that the sample information includes patient information and the like. At present, when the oocyte or the embryo is frozen, the sample information is adhered to the mark section 220 of the carrying rod 200 in a sticker mode or handwritten on the mark section 220 by a marker pen, the mode is simple to operate, but information omission or errors are easy to occur in the freezing process, for example, information is fuzzy due to label falling and artificial friction, and the traceability of the sample information is not facilitated; meanwhile, in liquid nitrogen or in a liquid nitrogen gasification environment, specific sample information cannot be seen clearly, samples need to be taken out one by one for confirmation, the risk of re-melting of the samples is increased, and batch storage and management in a sample library cannot be achieved.

Therefore, the marking section 220 is preferably provided with an information identification electronic tag for storing the sample information, so that the sample information in the information identification electronic tag can be obtained in a non-contact manner. Further, the information identification electronic tag includes at least one of a bar code, a two-dimensional code, and an electronic chip tag.

Further, the preferred information identification electronic tag is an electronic chip tag, more preferably a Radio Frequency Identification (RFID) electronic chip tag, and can read or write in sample information through a radio frequency technology, and the electronic chip tag can be embedded or mounted on the carrier rod 200, so that the electronic chip tag can be stored for a long time under liquid nitrogen, and cannot be damaged due to the influence of thermal expansion and cold contraction when entering and exiting a liquid nitrogen environment; in addition, the method can still accurately acquire sample information in liquid nitrogen or in a liquid nitrogen gasification environment, and the quality of other samples is not influenced.

The grip section 210 of the conventional slide bar 200 is mostly smooth, and is easy to slide and difficult to control during operation. Referring to fig. 6, in some embodiments of the present invention, it is preferable that the grip section 210 of the slide bar 200 is provided with an anti-slip texture 211. Further, the length of the anti-slip texture 211 is at least 2/3 of the entire grip section 210.

The conventional carrier bar 200 has a fixed length and is inconvenient to use. During the use of the vitrified frozen carrier 10, the convenience and safety of use in different occasions need to be adapted, for example, when a sample is loaded under a microscope, the holding section 210 of the carrying rod 200 is required to have a moderate length, which is convenient for the stable control of the handle and the accurate loading of the sample; when liquid nitrogen is inserted for vitrification freezing, the holding section 210 of the carrying rod 200 is required to be elongated in length to prevent hands from being injured by accidental freezing; the holding section 210 of the carrier rod 200 is required to be shortened in length for storage and management in the cell bank to save storage space in the liquid nitrogen tank. Accordingly, in some embodiments of the present invention, it is preferred that the gripping section 210 of the carrier rod 200 be provided in a length adjustable configuration.

In some specific examples, the gripping section 210 of the carrier bar 200 is a telescopic bar (as shown in fig. 7) or a corrugated telescopic bar (as shown in fig. 8).

Referring to fig. 7, further, the telescopic rod includes a plurality of rods 212, and the rods 212 are sequentially sleeved and connected with each other, and two rods 212 are connected at one end. Two loop bars 212 at the ends are connected to the slide 100 and the marking segments 220, respectively.

Referring to fig. 8, the bellows includes a bellows portion 213 and two fixing portions 214 disposed at two ends of the bellows portion 213, and the two fixing portions 214 are respectively connected to the slide 100 and the marking section 220.

An embodiment of the present invention provides a vitrification cryopreservation method using any one of the above vitrification freezing vehicles, including the following steps S10 to S30:

s10: transferring the sample treated by the vitrified refrigerating fluid into a groove of a slide glass, and absorbing redundant liquid in the groove;

s20: placing a sealing piece on a slide glass with a sample, and attaching the sealing piece to the slide glass to seal the groove;

s30: and (4) holding the carrying rod, and transferring the vitrified frozen carrier with the configured sample into liquid nitrogen for preservation.

According to the vitrification freezing storage method, any vitrification freezing carrier is adopted, the operation process is simpler and more stable, the efficiency of transferring oocyte or embryo samples is higher, the problem of cross contamination of a plurality of samples stored in liquid nitrogen is effectively solved, and the vitrification freezing reliability is improved.

In some of these embodiments, before the step of transferring the sample into the recess, the following step is further included: and presetting the sample information into the information identification electronic tag.

In some embodiments, before the step of holding the slide bar to transfer the vitrified frozen carrier with the configured sample to liquid nitrogen for preservation, the method further comprises the following steps: the length of the holding section of the carrying rod is adjusted.

In order to reduce the osmotic damage and the toxic damage of cells in the vitrified refrigerating fluid, the vitrified refrigerating fluid is loaded by adopting a step method at present, but the transfer steps are not as many as possible, because the oocytes and/or embryos are easily lost due to the transfer of a plurality of times. At present, three-step method loading is basically adopted in the market, so that the operation is complex on one hand, the risk of cell loss is increased, and on the other hand, the overlong balance time of cells in a solution is not beneficial to batch processing of samples such as oocytes or embryos clinically. Meanwhile, the formula of the vitrified refrigerating fluid is too simple, so that the freezing damage of cells can not be well reduced, and a good low-temperature preservation effect is achieved.

In some embodiments of the present invention, the vitrified cooling fluid comprises a first cooling fluid and a second cooling fluid; the first refrigerating fluid comprises a base fluid and a permeability protective agent, and the second refrigerating fluid comprises the base fluid, the permeability protective agent and a non-permeability protective agent.

Further, the permeability protective agent in the first refrigerating fluid and the second refrigerating fluid comprises glycol and dimethyl sulfoxide; the non-osmotic protective agent includes sucrose. The base liquid in the first refrigerating liquid and the second refrigerating liquid comprises TCM199, 10 mM-20 mM HEPES and 0.1% -0.5% (w/w) human serum albumin, or DMEM, 10 mM-20 mM HEPES and 0.1% -0.5% (w/w) human serum albumin.

Further, the first refrigerating fluid comprises 5% -8% (v/v) Ethylene Glycol (EG), 5% -8% (v/v) dimethyl sulfoxide (DMSO) and 84% -90% (v/v) base fluid.

Further, the second refrigerating fluid comprises 12-20% (v/v) Ethylene Glycol (EG), 12-20% (v/v) dimethyl sulfoxide (DMSO), 0.5-1.5M cane sugar and 60-76% (v/v) base fluid.

Further, the sample is sequentially processed by adopting a first refrigerating fluid and a second refrigerating fluid, and the balance time of the sample in the first refrigerating fluid and the balance time of the sample in the second refrigerating fluid are respectively 3 min-5 min and 30 s-60 s.

According to the utility model, by optimizing the composition of the vitrified refrigerating fluid and combining the first refrigerating fluid and the second refrigerating fluid, the equilibrium time is shortened, and meanwhile, the intracellular moisture can be reduced to the maximum extent, so that the vitrified preservation effect is improved.

In order to make the objects, technical solutions and advantages of the present invention more concise and clear, the present invention is described with the following specific embodiments, but the present invention is by no means limited to these embodiments. The following described examples are only preferred embodiments of the present invention, which can be used to describe the present invention and should not be construed as limiting the scope of the present invention. It should be understood that any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

The cooling rate performance test and the vitrification cryopreservation effect test are performed on the freezing carrier and the comparison product.

Cooling rate performance test

The freezing carrier and the control product, namely an imported product (with the model specification of 2013), in the utility model shown in the figure 1 are measured according to a method mentioned in the literature "measurement of the cooling rate of the freezing carrier Cryotop and system optimization" (2013)

The structure of which is shown in fig. 9) was measured for the cooling rate during use, and the result is shown in fig. 10. As can be seen from FIG. 10, the cooling rate of the freezing carrier of the present invention is significantly higher than that of the control product, which indicates that the reduction in the thickness of the slide 100 at the portion (groove) in contact with the embryo can significantly increase the cooling rate, and is more conducive to the vitrification preservation of the embryo.

(II) testing vitrification cryopreservation effect

Example 1

The vitrification freezing carrier shown in figure 1 and the following first freezing liquid and second freezing liquid are adopted to carry out vitrification freezing preservation on the mouse cell embryo.

The first freezing fluid comprises 7.5% (v/v) Ethylene Glycol (EG), 7.5% (v/v) dimethyl sulfoxide (DMSO) and 85% (v/v) base fluid.

The second freezing fluid comprises 15% (v/v) Ethylene Glycol (EG), 15% (v/v) dimethyl sulfoxide (DMSO), 1.0M sucrose and 70% (v/v) base fluid.

Wherein the basic solution comprises TCM199 culture medium or DMEM culture medium, 10 mM-20 mM HEPES (4-hydroxyethyl piperazine ethanesulfonic acid) and 0.1% -0.5% (w/w) Human Serum Albumin (HSA).

Taking mouse 1-cell embryo and 2-cell embryo (2-cell embryo is cell embryo at later stage in the growth process of 1-cell embryo, and the two stages are the stages that cells are easy to be injured in the freezing process), the concrete operation method of the vitrification cryopreservation of mouse cell embryo is as follows:

(1) embedding sample information of mouse embryos to Radio Frequency Identification (RFID) electronic chip tags of the marker segments 220;

(2) the marking section 220, the holding section 210 and the slide 100 are connected into a slide whole with a carrying rod, the length of the holding section 210 (telescopic rod of the sleeve) is adjusted to be proper, and the slide is put into liquid nitrogen for precooling for later use;

(3) cleaning mouse 1-cell embryo and 2-cell embryo, transferring into first refrigerating fluid, and balancing for 3-5 min; then transferring the mixture into a second refrigerating fluid, and balancing for 30-60 s;

(4) placing the vitrified carrier (carrying rod slide whole) without the sealing sheet 300 under a microscope, transferring the balanced 1-cell embryo into a groove 101 on a slide 100, wherein each groove 101 at least has one cell and absorbs redundant liquid but does not absorb the cell;

(5) placing the sealing sheet 300 on the slide 100 with cells, and sealing the groove 101 by hot pressing;

(6) adjusting the length of the holding section 210, holding the holding section 210 by hand, and quickly inserting the vitrified frozen carrier 10 containing 1-cell embryos into liquid nitrogen;

(7) the length of the holding section 210 is adjusted to fix the vitrified frozen carrier 10, which has been vitrified frozen, on a special rack for long-term storage in liquid nitrogen.

Blank control group

Blank group means that embryo is not treated, i.e. not treated with vitrification refrigerating fluid/thawing fluid and frozen by liquid nitrogen.

Comparative example 1

Comparative example 1 is essentially the same as the vitrification freezing process of example 1, except that: the vitrified frozen carrier in example 1 was replaced with an imported product of the type and specification

The specific structure is shown in the figure below, the embryo freezing surface is of a flat plate type, the requirement on an operator is high, the problems of cell sliding or large liquid remaining amount and the like are easy to occur, and the efficiency is low.

Further, the embryos were subjected to vitrification cryopreservation for one week according to the methods of example 1 and comparative example 1, and then the cryopreserved embryos were thawed, eluted with a protectant, re-cultured, and the final blastocyst rates thereof were observed. Specific results are shown in table 1. As can be seen from the table, the 1-cell blastocyst rate and the 2-cell blastocyst rate in example 1 are significantly higher than those in comparative example 1, which shows that the freezing carrier and freezing fluid set in the application has a significant advantage on the vitrification cryopreservation effect of the embryo.

TABLE 1

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the utility model. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims, and the description and the drawings can be used for explaining the contents of the claims.

Claims

1. A vitrified frozen carrier comprising:

a carrier bar; the carrying rod comprises a holding section and a marking section connected with the holding section, the marking section is used for marking sample information, and the length of the holding section of the carrying rod is adjustable;

the slide glass is arranged at one end of the carrying rod, a groove is formed in the slide glass, and the carrying rod is connected with the slide glass through one end where the holding section is located; and

and the sealing piece is attached to the slide glass to seal the groove.

2. The vitrification freezing carrier of claim 1 wherein the slide comprises a sheet body region and a recessed region, the recess being disposed in the recessed region;

3. A vitrified frozen carrier according to claim 2 wherein the slide has a thickness in the region of the recess of between 50 μm and 200 μm; the thickness of the carrier sheet in the area of the sheet body is 200-500 mu m.

4. The vitrification freezing carrier of claim 1 wherein the slide comprises a sheet body region and a recessed region, the recess being disposed in the recessed region; the depth of the groove is greater than the thickness of the bulk region of the sheet.

5. A vitrified frozen carrier according to claim 1 wherein the groove has a radial dimension of the mouth of the groove of between 100 μm and 200 μm; and/or

The depth of the groove is 150-200 μm.

6. A vitrified frozen carrier as claimed in claim 1, characterized in that the recesses are cylindrical or prismatic; and/or

The bottom wall of the groove is a plane or a concave cambered surface.

7. A vitrified frozen carrier according to claim 1 wherein the recesses in the slide are plural and the sealing plate is used to seal each of the recesses in the slide.

8. The vitrified frozen carrier of claim 1 wherein the sealing sheet is a transparent sealing sheet; and/or

The thickness of the sealing piece is 50-100 mu m.

9. The vitrified frozen carrier of claim 1 wherein the marking segment is provided with an information-identifying electronic tag for storing sample information.

10. The vitrified frozen carrier of claim 9 wherein the information-identifying electronic label comprises at least one of a bar code, a two-dimensional code, and an electronic chip label.

11. The vitrification freezing carrier of claim 1 wherein the holding section of the carrier bar is a telescopic bar comprising a plurality of bars, the bars are sequentially sleeved and connected with each other, two bars are connected at one end, and two bars at the end are connected with the slide and the marking section respectively; or,

the holding section of the carrying rod is a corrugated telescopic rod, the corrugated telescopic rod comprises a corrugated telescopic part and fixing parts arranged at two ends of the corrugated telescopic part, and the two fixing parts are respectively connected with the carrying sheet and the marking section.

12. The vitrified frozen carrier of claim 1 wherein the gripping section of the carrier bar is provided with a non-slip texture having a length of at least 2/3 of the entire gripping section.