CN113558041A

CN113558041A - Ovary tissue freezing method based on freezing carrier for ovary tissue freezing

Info

Publication number: CN113558041A
Application number: CN202110665091.0A
Authority: CN
Inventors: 马超
Original assignee: Shanghai Huanyi Medical Technology Co ltd
Current assignee: Shanghai Huanyi Medical Technology Co ltd
Priority date: 2021-06-16
Filing date: 2021-06-16
Publication date: 2021-10-29

Abstract

The invention discloses an ovarian tissue freezing method based on a freezing carrier for ovarian tissue freezing, which adopts a clip-type fixing device design, avoids using tweezers to clamp tissue slices one by one in the freezing and unfreezing processes, can complete the transfer of the tissue slices by holding a freezing inner tube by hand, greatly reduces the labor intensity of operators, and reduces the risks of freezing solution concentration change and pollution caused by repeated use of the tweezers. The design of the vertical insertion type freezing and thawing dish is increased, the tissue piece is in freezing liquid and thawing liquid, and the tissue piece is fixed on the inner pipe, the freezing inner pipe can be clamped on the clamping groove of the freezing and thawing dish, so that the floating phenomenon of the tissue piece is avoided, and the freezing and thawing effect is fully ensured. The design of the closed type freezing outer tube isolates the direct contact of liquid nitrogen and tissue slices in a heating and sealing mode, and the pollution risk is reduced. The freezing inner tube is convenient to fix due to the design of the clamping groove in the freezing outer tube, and the operation stability is ensured.

Description

Ovary tissue freezing method based on freezing carrier for ovary tissue freezing

Technical Field

The invention relates to a tool used in combination with tissue freezing and a using method thereof, in particular to a tool and a method used in ovarian tissue freezing preservation technology.

Background

Ovarian tissue cryopreservation techniques are the fastest and most effective method of preserving fertility for adult women to be treated with chemoradiotherapy, and for prepubertal women, ovarian tissue freezing is the only way to preserve fertility.

At present, the ovarian tissue cryopreservation has no unified standard, and the common cryopreservation method comprises slow programmed freezing and vitrification freezing. The traditional slow programmed freezing is difficult to popularize clinically due to the defects of high price, long freezing time, complex operation and the like of instruments. The ovary tissue is vitrified and frozen, so that the ovary tissue can be in a vitrified state in a very fast cooling process and a very short time, the formation of ice crystals in cells is avoided, and the method has the characteristics of rapidness, high efficiency, simplicity, convenience and economy and is widely applied.

The freezing carrier is one of the key factors for the success of the ovarian tissue vitrification freezing. The vitrification freezing carrier used for freezing the ovary tissue at present mainly comprises: frozen straws, frozen rings, cell screens, needle punched carriers, and KITAZATO commercial carriers, among others, wherein frozen straws and frozen rings can only be loaded with a small volume of ovarian tissue; when the cell screen is placed in a liquid nitrogen tank for storage, the occupied volume is too large, and the cell screen is not easy to store in batches; the needled carrier can cause irreversible mechanical damage to ovarian tissue; the KitaZATO company commercialized carrier has small contact area with ovarian tissues, is easy to fall off, and is difficult to popularize in clinical and scientific research works.

In addition, in the traditional ovarian tissue vitrification freezing process, the following problems often exist:

1. the operator needs to cut a large ovarian tissue obtained by surgery into several to several tens of tissue pieces with a size of 10mm x 10mm x 1mm, which are different from each other, with medical forceps. An operator needs to clamp and move each ovarian tissue by using medical forceps in the whole process of the vitrification freezing of the ovarian tissue so as to achieve the purpose of dehydrating the ovarian tissue in the cryoprotectant with different concentrations, and the labor intensity of the operator is greatly increased in the process.

2. When entering the refrigerating fluid, the tissue slices can float on the refrigerating fluid surface due to buoyancy, and the floating tissue slices are continuously pressed by tweezers to be immersed in the refrigerating fluid, so that the tissue is fully dehydrated, and the refrigerating effect is achieved. The process is complicated, and the labor intensity of an operator is greatly increased.

3. In the freezing process, when the medical forceps are used for transferring tissue slices between freezing liquids with different concentrations, the following two problems exist. Firstly when transferring to high concentration refrigerating fluid from low concentration, medical tweezers touched high concentration refrigerating fluid very easily, must be with tweezers clean up this moment, when otherwise carrying out this operation once more, can bring part high concentration liquid into among the low concentration liquid, influence refrigerating fluid composition and concentration, and then influence freezing effect. Secondly, when the tissue slices are transferred by two refrigerating fluids with different concentrations, the tissue slices need to be transferred repeatedly by using tweezers, so that the risk of pollution of the tweezers is greatly increased, and the tissue slices are further polluted.

4. The traditional ovary tissue freezing mode is an open type, and ovary tissue slices are directly contacted with liquid nitrogen, so that the risk of cross contamination among the tissue slices is increased.

In conclusion, the closed ovarian tissue freezing carrier which is simple and convenient to operate, safe and reliable to store, non-toxic and pollution-free is urgently needed to overcome a great deal of inconvenience in clinic.

Disclosure of Invention

Aiming at the defects of the ovarian tissue vitrification freezing process, the invention provides a closed ovarian tissue freezing carrier which is simple and convenient to operate, safe and reliable to store, compatible, ordered, non-toxic and pollution-free, and a method for using the freezing carrier. Aims to achieve the purposes of convenient operation and improving the operation applicability.

In order to achieve the above object, the present invention provides a clamping device for a frozen carrier, comprising an inner clamping ring and an outer clamping ring which work in a clip manner and are used for clamping tissue slices, and a handle which is connected with one side of the inner clamping ring and one side of the outer clamping ring.

In a preferable mode, the inner clamping ring and the outer clamping ring are both made of 304 stainless steel, the width of the outer clamping ring is 7-8mm, and the length of the outer clamping ring is 20 mm; the width of the inner clamping ring is 4-5mm, and the length of the inner clamping ring is 15-18 mm; the handle is made of nontoxic medical plastic; the diameter of the steel wire of the inner clamping ring and the outer clamping ring is 0.1-0.2mm,

the invention also provides a freezing outer tube for freezing the carrier, and the inner wall of the tube body in the form of a test tube is correspondingly provided with clamping grooves in the axial direction of the tube body at two radial sides.

In a preferable mode, the freezing carrier is made of a freezing outer tube which is made of a medical nontoxic PVC hose, the width of the inside of the freezing outer tube is 12mm, and the length of the freezing outer tube is 25-30 mm; the width of the clamping groove is slightly wider than the diameter of the clamped object.

The invention provides a frozen carrier method for realizing tissue freezing by using the tool, which is characterized by comprising a freezing process and a thawing process, wherein the freezing process comprises the following steps:

s1, cutting tissue slices with the size of 10mm x 10mm x 1mm into slices by medical forceps, and sequentially fixing the slices on a clip type device; the tissue slice is arranged above an outer clamping ring 1 of the clamping device, below an inner clamping ring 2 of the clamping device, a handle 3 of the clamping device is held by hands, and the clamping device with the tissue slice clamped is vertically inserted into a freezing dish clamping groove containing ES liquid to be balanced for 25 minutes;

s2, holding the handle 3 of the clamping device by hand, fishing out the clamping device from the ES solution, adsorbing redundant ES solution on the tissue slice by using sterile filter paper, and then vertically inserting the clamping device into a clamping groove of a freezing dish containing VS solution in sequence for balancing for 15 minutes;

s3, holding the handle 3 of the clamping device by hand, fishing out the clamping device from the VS liquid in sequence, adsorbing redundant VS liquid on the tissue slice by using sterile filter paper, inserting the tissue slice into the freezing outer tube with the clamping groove, just inserting the tissue slice into the clamping groove of the outer tube, and putting the tissue slice into liquid nitrogen for storage after heat-sealing the mouth of the freezing outer tube by using a heater.

The thawing process comprises the following steps:

s4, in liquid nitrogen, using medical forceps to clamp the freezing outer tube by one hand, carefully exposing the heat-sealed part of the freezing outer tube out of the liquid nitrogen, using medical scissors to cut the heat-sealed part of the freezing outer tube by the other hand, quickly taking out the clamping device in the freezing outer tube, directly inserting the clamping device into a clamping groove of an inner tube of a thawing dish containing TS liquid and pre-warmed to 37 ℃, and keeping the clamping device for 60 seconds;

s5, sequentially passing the mixture in a thawing dish containing DS liquid for 3 minutes, a thawing dish containing WS1 liquid for 5 minutes and a thawing dish containing WS2 liquid for 5 minutes at room temperature according to the above operation; and then taking the recovered tissue piece off the clamping device, putting the recovered tissue piece into a tissue culture solution, culturing at 37 ℃ for 2 hours, and then transplanting.

The invention has the beneficial effects that:

1. clip centre gripping formula fixing device (freezing inner tube) design has avoided using tweezers to press from both sides one by one and has got the tissue piece at the freezing in-process that unfreezes, and handheld freezing inner tube can accomplish the transfer of tissue piece, greatly reduced operator's intensity of labour to reduced because tweezers use repeatedly and caused the risk that refrigerating fluid concentration changes and pollutes.

2. The design of the vertical insertion type freezing and thawing vessel (the clamping groove of the built-in inner tube) is added, the tissue piece is in the freezing liquid and the thawing liquid, the tissue piece is fixed on the inner tube, the freezing inner tube can be clamped on the clamping groove of the freezing and thawing vessel, the floating phenomenon of the tissue piece is avoided, and the freezing and thawing effect is fully guaranteed.

3. The design of the closed type freezing outer tube isolates the direct contact of liquid nitrogen and tissue slices in a heating and sealing mode, and the pollution risk is reduced. The freezing inner tube is convenient to fix due to the design of the clamping groove in the freezing outer tube, and the operation stability is ensured.

Drawings

Fig. 1 is a schematic structural view of a clamp-type device for freezing a carrier according to the present invention.

Fig. 2 is a schematic view of the clamping device shown in fig. 1 in a use state.

FIG. 3 is a schematic diagram of the construction of the freeze and thaw tray during use of the invention.

Fig. 4 is a schematic view showing a state of use of the freezing and thawing tray shown in fig. 3.

Fig. 5 is a schematic view of the structure of the freezing outer tube.

Fig. 6 is a schematic view showing a state of use of the freezing outer tube shown in fig. 3.

Detailed Description

As shown in fig. 1 and 2, the present invention firstly provides a clip-type device for freezing carrier, i.e. clip-type fixing device: the inner tube is also called as freezing inner tube, which is composed of an inner clamping ring, an outer clamping ring and a handle part, wherein the inner clamping ring and the outer clamping ring are both made of 304 stainless steel, the inner diameter of the clamping ring is 0.1-0.2mm, and the handle is made of nontoxic medical plastic. The width of the outer clamping ring is 7-8mm (slightly smaller than the diameter of the ovarian tissue slice by 10mm), and the length of the outer clamping ring is 20 mm. The width of the inner clamping ring is 4-5mm, and the length of the inner clamping ring is 15-18 mm.

As shown in fig. 5 and 6, the present invention also provides an insertion type tissue freezing and heat sealing tube: also called as a freezing outer tube, the freezing outer tube is made of a medical nontoxic PVC hose. The width of the freezing outer tube is 12mm, and the length of the freezing outer tube is 25-30 mm. The clamping grooves of the freezing inner tube are arranged in the freezing inner tube, and the distance between the clamping grooves is smaller than the width of the outer clamping ring of the freezing inner tube. The heat sealing area is positioned at the mouth of the freezing outer tube and can be sealed after being heated by a heat sealing device.

As shown in figures 3 and 4, the invention adopts the vertical insertion type freezing and thawing dish and the thawing dish (a plurality of inner tube clamping grooves are arranged inside and made of medical nontoxic plastic materials) in the prior art.

The working process of the invention comprises a freezing process and a thawing process.

The freezing process comprises the following steps:

1. tissue sections cut to a size of 10mm x 10mm x 1mm were sequentially fixed to a clip-type clip fixing device (hereinafter, the frozen inner tube was used instead) with medical forceps. The tissue piece is positioned above the outer clamping ring and below the inner clamping ring and is fixed by the inner clamping ring and the outer clamping ring (as shown in figures 1 and 2). The handle portion of the inner tube was held by hand and the inner tube was subsequently inserted upright into a freezing dish well containing ES liquid (equilibration liquid) and equilibrated for 25 minutes (see fig. 3 and 4).

2. And (3) holding the handle part of the freezing inner tube again, fishing out the freezing inner tube from the ES liquid, adsorbing redundant ES liquid on the tissue piece by using sterile filter paper, and vertically inserting the freezing inner tube into a clamping groove of a freezing dish containing VS liquid (vitrified refrigerating liquid) in sequence for balancing for 15 minutes.

3. After the balance time is finished, the handle part of the freezing inner tube is held by hands, the freezing inner tube is sequentially fished out from the VS liquid, and is inserted into the freezing outer tube after the unnecessary VS liquid on the tissue piece is adsorbed by sterile filter paper (a clamping groove is designed in the freezing outer tube, and the freezing fixing device just can be inserted into the clamping groove of the outer tube), and the mouth of the freezing outer tube is heat-sealed by a heater and then is put into liquid nitrogen for storage (as shown in fig. 5 and 6).

The thawing process comprises the following steps:

1. in liquid nitrogen, the freezing outer tube is clamped by medical forceps with the left hand, the heat-sealed part of the freezing outer tube is carefully exposed out of the liquid nitrogen, the heat-sealed part of the freezing outer tube is cut off by medical scissors with the right hand, the freezing inner tube in the freezing outer tube is quickly taken out and directly inserted into a clamping groove of an inner tube of a thawing dish which contains TS (thawing solution) and is pre-warmed to 37 ℃ for 60 seconds.

2. According to the above procedure, the mixture was passed through a thawing dish containing DS solution (diluent) for 3 minutes, a thawing dish containing WS1 solution (washing solution 1) for 5 minutes, and a thawing dish containing WS2 solution (washing solution 2) for 5 minutes at room temperature. And then taking the recovered tissue piece down from the clip-type fixing device of the clip, putting the tissue piece into a tissue culture solution, culturing at 37 ℃ for 2 hours, and then transplanting.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.

Claims

1. A method for freezing ovarian tissue based on a freezing carrier for freezing ovarian tissue is characterized in that,

a setting tool: the clamping type device for freezing the ovarian tissue comprises an inner clamping ring (2) and an outer clamping ring (1) which work in a clip mode of a clip needle and are used for clamping tissue slices, and a handle (3) which is connected with one side of the inner clamping ring (2) and one side of the outer clamping ring (1) together;

a setting tool: the freezing outer tube for freezing the ovarian tissue is characterized in that the inner wall of the tube body in the form of a test tube is correspondingly provided with clamping grooves in the axial direction of the tube body at two radial sides, and the upper part of the tube body is provided with a heat sealing area;

the freezing process comprises the following steps:

s1, cutting tissue slices with the size of 10mm x 10mm x 1mm into slices by medical forceps, and sequentially fixing the slices on a clip type device; the tissue slice is arranged above an outer clamping ring (1) of the clamping device, a handle (3) of the clamping device is held by a hand below an inner clamping ring (2) of the clamping device, and the clamping device with the tissue slice clamped is inserted into a freezing dish clamping groove containing ES liquid to balance for 25 minutes in sequence;

s2, holding a handle (3) of the clamping device by hand, fishing out the clamping device from the ES solution, adsorbing redundant ES solution on the tissue slice by using sterile filter paper, and then vertically inserting the clamping device into a clamping groove of a freezing dish containing VS solution in sequence for balancing for 15 minutes;

s3, holding a handle (3) of the clamping device by hand, fishing out the clamping device from the VS liquid in sequence, adsorbing redundant VS liquid on the tissue slice by using sterile filter paper, inserting the tissue slice into a freezing outer tube with a clamping groove, just inserting the tissue slice into the clamping groove of the outer tube, and putting the tissue slice into liquid nitrogen for storage after heat-sealing the mouth of the freezing outer tube by using a heater.

2. The method for freezing ovarian tissue as claimed in claim 1, wherein the thawing process comprises the following steps:

3. The ovarian tissue freezing method according to claim 1 or 2, wherein the inner clamping ring (2) and the outer clamping ring (1) of the clamping device are both made of 304 stainless steel, and the width of the outer clamping ring (1) is 7-8mm, and the length of the outer clamping ring is 20 mm; the width of the inner clamping ring (2) is 4-5mm, and the length is 15-18 mm; the handle is made of nontoxic medical plastic; the diameter of the steel wire of the inner clamping ring (2) and the outer clamping ring (1) is 0.1-0.2 mm.

4. The ovarian tissue freezing method according to claim 1 or 2, wherein the freezing outer tube is made of medical nontoxic PVC soft tube, the width of the freezing outer tube is 12mm, and the length of the freezing outer tube is 25-30 mm; the width of the clamping groove is slightly wider than the diameter of the clamped object.