CN101314757A - Aculeus type tissue slice glassivation refrigeration carrier and refrigeration method for ovary tissue - Google Patents
Aculeus type tissue slice glassivation refrigeration carrier and refrigeration method for ovary tissue Download PDFInfo
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- CN101314757A CN101314757A CNA200810045568XA CN200810045568A CN101314757A CN 101314757 A CN101314757 A CN 101314757A CN A200810045568X A CNA200810045568X A CN A200810045568XA CN 200810045568 A CN200810045568 A CN 200810045568A CN 101314757 A CN101314757 A CN 101314757A
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Abstract
An acupuncture-type tissue slice vitrification freezing carrier is composed of a freezing tube with one end being closed and the other end being open, and an acupuncture device. The acupuncture device comprises a needle head and a needle disposed on the needle head and used for threading tissue slices. A section matching with the internal shape and size of the open end of the freezing tube is disposed on the needle head. After the acupuncture device and the freezing tube are combined, the needle is positioned in the freezing tube, and the needle head is adopted as a tube plug of the freezing tube to seal the open end of the freezing tube. A plurality of tissue slices (such as ovary tissue slices and liver tissue slices) can be threaded in a row on the needle at intervals by the carrier so as to be subjected to each operation of the vitrification freezing and storage, and a plurality of sample tissues can be subjected to the vitrification freezing and storage simultaneously, with simple operation and high efficiency, so that the sample tissues can be dehydrated and refrigerated under the same conditions. The storage of the ovary multicellular ingredients (such as the storage of interstitial cells and extracellular matrix ingredients) is superior to the programmed slow freezing and the carrier-free vitrification freezing method.
Description
Technical field
The invention belongs to glass freezing and preserve field, the particularly vitrified frozen vector of organ or tissue such as ovary, liver and freezing method.
Background technology
Along with profound hypothermia is preserved development of technology, Refrigeration Technique has been not limited to frozen cell, gamete or embryo.To the frozen of histoorgan is the development trend of Refrigeration Technique.Vitrifying freeze process is the supper-fast freezing method that proposes in recent years; than the freezing weak point consuming time of sequencing; convenient cheap; no ice crystal damage; research think be more suitable for the not single tissue of cellular constituent or organ frozen; but the shortcoming that glass freezing exists is: required cryoprotection agent concentration is higher, causes cytotoxicity to increase, and has limited its application in tissue, organ are preserved.Improving freezing carrier or method and improve freezing rate to reduce the concentration of cryoprotectant, is scholar's main contents that research is explored to glass freezing in recent years.
Ovary is important reproduction of women and endocrine organ, and it contains the cellular constituent of multiple different qualities, and ovocyte has non-renewable characteristic, and therefore the freezing preservation for ovary has more difficulty and challenge than other histoorgan.Some the treatment of malignant tumour survivor, autoimmune disease of heavy dose of chemoradiotherapy and other iatrogenic factors have been experienced and patient that Premature Ovarian Failure takes place, the ovary cortex tissue that takes a morsel before the disease treatment carries out freezing preservation, the recovery of thawing is afterwards transplanted, recovering menstruation and fertility brings hope to the patient, is the focus and the difficult point problem of present reproductive medicine research field.
Mammalian Ovary is being organized in the glass freezing; traditional vitrification method is to take freeze pipe as carrier (straw or freezing tubule); ovary tissue is cut into sheet or strip; after adopting the cryoprotectant dehydration of high density, will organize suck or put into freeze pipe and seal together with protective material after, directly put into liquid nitrogen again; this method cryoprotectant volume is bigger; rate of temperature fall is slower, therefore, is replaced by the freezing method that directly contacts with liquid nitrogen in recent years.In freezing research to mouse and people's ovary tissue, adopt polyester fiber film or Electronic Speculum copper mesh (to see HasegawaA as carrier, Hamada Y, Mehandjiev T and Koyama K (2004) In vitro growth and maturation as well asfertilization of mouse preantral oocytes from vitrified ovaries.Fertil Steril 81,824-830.; Isachenko E, Isachenko V, Rahimi G and Nawroth F (2003) Cryopreservation of humanovarian tissue by direct plunging into liquid nitrogen.Eur J Obstet Gynecol Reprod Biol108,186-193.); Same principle has research to adopt nylon wire as freezing carrier; Perhaps ox or mouse ovarian tissue block are put into freeze pipe, the freeze pipe of open-ended is placed directly in the liquid nitrogen or a small amount of liquid nitrogen directly is covered in ovary tissue increases tissue and liquid nitrogen contact area, (see Bordes A to accelerate rate of temperature fall, Lornage J, Demirci B, FranckM, Courbiere B, Guerin JF and Salle B (2005) Normal gestations and live births afterorthotopic autograft of vitrified-warmed hemi-ovaries into ewes.Hum Reprod 20,2745-2748.; Chen SU, Chien CL, Wu MY, Chen TH, Lai SM, Lin CW and Yang YS (2006) Novel direct cover vitrification for cryopreservation of ovarian tissues increases follicleviability and pregnancy capability in mice.Hum Reprod 21,2794-2800); In addition, also there is research to adopt DNAcarrier free glass freezing (to see Yeoman RR, Wolf DP and Lee DM (2005) Coculture ofmonkey ovarian tissue increases survival after vitrification and slow-ratefreezing.Fertil Steril 83,1248-1254; Li YB, Zhou CQ, Yang GF, Wang Q and DongY (2007) Modified vitrification method for cryopreservation of human ovarian tissues.ChinMed J (Engl) 120,110-114).These methods or can not make fully contact liquid nitrogen of tissue, perhaps the residual freezing rate, particularly multiple sample of can reducing of the cryoprotectant of tissue surface carries out can increasing operation easier when freezing simultaneously, is difficult to guarantee that each sample is with same condition dehydration and freezing.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art; provide a kind of novel aculeus type tissue slice glassivation freezing carrier to reach and the supporting freezing method of this carrier; use this kind carrier and supporting freezing method; not only can improve rate of temperature fall; reduce cryoprotection agent concentration and toxicity; carry out simultaneously when freezing at multiple sample, make the various kinds instinct with same condition dehydration and freezing, and simple to operate, efficient.
Aculeus type tissue slice glassivation freezing carrier of the present invention by an end seal, the frozen pipe and the needling device of an end opening form; Described needling device comprises syringe needle and is installed in being used on the syringe needle wears the pin of tissue, and syringe needle is provided with and the interior shape of frozen tube opening end and portion's section of size match; After the combination of needling device and frozen pipe, the pin that is used to wear tissue is positioned at frozen pipe, and syringe needle is that the pipe close of frozen pipe seals frozen tube opening end.
For preventing that carrier is put in the pressure cataclysm that causes because of the liquid nitrogen volatilization in the air and makes frozen pipe explosive, be provided with dividing plate in the frozen pipe, space between this dividing plate and the opening end is used to adorn liquid nitrogen and the tissue that is through on the pin, the decompression filler is equipped with in the space that this dividing plate and blind end and the tube wall between them surround, and the closed end wall of dividing plate, frozen pipe is or/and have vent on the tube wall between them.The preferred version of dividing plate installation site: the spacing L1 of dividing plate and frozen duct occlusion end be dividing plate and frozen tube opening end spacing L2 1/5th to 1/4th.The decompression filler is cotton cord or flexible materials such as cotton yarn or other fiber.
The pin that is used to wear tissue is at least one (being one or more), avoids the damage organized for knowing clearly, and the diameter of described pin is not more than 0.2mm, preferred 0.12～0.18mm.
For the ease of operation, needling device can be provided with handle, and described handle is connected with syringe needle.In order both to be convenient to operation, save the storage area again, the present invention becomes long handle and short handle with handle design, and long handle and short handle can be integral structures, the contraction structure that is easy to fracture in the boundary setting of long handle and short handle; Long handle and short handle also can be two members, adopt plug-in to connect.Put at the carrier that liquid nitrogen will be housed and be frozen tissue like this and can remove long handle easily and quickly before liquid nitrogen container stores.
In the carrier of the present invention, bottle stopper or short handle can adopt distinct colors, are convenient to the grouping of sample; Bottle pipe sidewall and handle side wall can adopt low temperature resistant marking pen to mark, with the different samples of convenient differentiation.
Carrier of the present invention can be used for the glass freezing of multiple tissue such as ovary, liver.
The method of glass freezing ovary tissue of the present invention is used above-mentioned aculeus type tissue slice glassivation freezing carrier, and operation steps is as follows:
(1) the people's ovary cortex sheet that obtains of will performing the operation is divided into the ovary tissue sheet in the culture dish that fills specimen collection liquid, then at least 4 ovary tissue sheets is through on the pin with being separated with spacing mutually;
(2) pin that will be installed with the ovary tissue sheet is transferred in the cryoprotectant after the dehydration, organizes the cryoprotectant on sheet surface with sterilization water-absorbing material suction removal ovary, and the pin that will be installed with the ovary tissue sheet is again put into the container that fills liquid nitrogen;
(3) frozen pipe is put into the container that fills liquid nitrogen, and make the liquid nitrogen frozen pipe of packing into, the pin that will be installed with the ovary tissue sheet then in liquid nitrogen is put into frozen pipe, and syringe needle is as the opening end sealing of pipe close with frozen pipe;
(4) freezing carrier that liquid nitrogen and ovary tissue sheet are housed of step (3) assembling being transferred to liquid nitrogen container stores.If the handle that the aculeus type tissue slice glassivation freezing carrier is provided with is made up of long handle and short handle, then remove long handle after, the freezing carrier that liquid nitrogen and ovary tissue sheet are housed that step (3) is assembled is transferred to liquid nitrogen container and stores.
The present invention has following beneficial effect:
1, use carrier of the present invention, organize when carrying out glass freezing simultaneously at multiple sample, not only simple to operate, efficient, and can make each sample with same condition contact refrigeration protective material and liquid nitrogen.
2, since carrier of the present invention a plurality of ovary tissue sheets can be through with being separated with spacing mutually conspiring to create one on the pin and arrange and to carry out the operations and the storage of glass freezing, make a plurality of tissue with identical condition dehydration and freezing, prevent the freezing one by one frozen effect instability that causes owing to dewatering time is inconsistent of sample.
3, use carrier operation of the present invention; can reduce to the cryoprotectant volume of ovary tissue surface attachment minimum; effectively improve rate of temperature fall and reduce the residual disadvantageous effect of cryoprotectant; and make each position of ovary tissue sheet all can directly contact liquid nitrogen; present existing vitrification method still can not satisfy the requirement of organizing the comprehensive engagement liquid nitrogen and minimizing the cryoprotectant volume simultaneously.
4, use carrier of the present invention; because the obvious raising of glass freezing rate of temperature fall; can adopt the cryoprotectant of low concentration (for example to dewater; the human ovarian organizes glass freezing; can adopt inferior maple of present minimum concentration cryoprotectant 15% dimethyl and 15% ethylene glycol; v/v), thereby have the hypotoxic advantage of lower concentration protective material, possess potential applicability in clinical practice.
5, experiment shows, use carrier of the present invention and supporting vitrification method thereof, can well preserve the form and the vigor of ovarian follicle, and the preservation (as the preservation of mesenchymal cell and extracellular matrix components) of ovary many cells composition is better than sequencing freezes slowly and the carrier free vitrifying freeze process; The animal model of the allogeneic heterotopic transplantation by mouse freeze thawing ovary shows: the growth of ovarian graft dive and functional rehabilitation better than other freezing method preservation effects, with fresh group the most approaching.
6, carrier of the present invention makes operation and memory function more perfect frozen pipe and needling device organic assembling, uses convenient; Syringe needle and handle arrangement be convenient to puncture sample and refrigeration operation, the frozen phial plug of syringe needle double as both simplified the structure, and was convenient to deposit sample again and took out sample.
7, the designed pressure reduction structure of frozen pipe in the carrier of the present invention can prevent that carrier is put in the pressure cataclysm that causes because of the liquid nitrogen volatilization in the air and makes frozen pipe explosive, thereby use safer.
8, in the carrier of the present invention, be short handle and the long handle that is easy to remove with handle design, both handled easilies is convenient to again put at the carrier that liquid nitrogen will be housed and be frozen tissue and is removed long handle before liquid nitrogen container stores, and saves the storage area.
Description of drawings
Fig. 1 is a kind of structure diagram of aculeus type tissue slice glassivation freezing carrier of the present invention;
Fig. 2 is another structure diagram of aculeus type tissue slice glassivation freezing carrier of the present invention;
Fig. 3 is first kind of structure iron of needling device;
Fig. 4 is second kind of structure iron of needling device;
Fig. 5 is the third structure iron of needling device;
Fig. 6 is a kind of mounting means synoptic diagram of three pins on syringe needle among Fig. 5;
Fig. 7 is three pins another mounting means synoptic diagram on syringe needle among Fig. 5;
Fig. 8 is the 4th a kind of structure iron of needling device;
Fig. 9 is the side-view of Fig. 8;
Figure 10 is the 5th a kind of structure iron of needling device;
Figure 11 is a kind of mounting means synoptic diagram of four pins on syringe needle among Figure 10;
Figure 12 is to use carrier of the present invention the ovary tissue sheet to be carried out the operation chart of glass freezing.
Among the figure, the frozen pipe of 1-, 2-dividing plate, 3-vent, 4-decompression filler, 5-long handle, 6-short handle, 7-syringe needle, 8-pin, 9-fill container, 10-long handle tweezer, the 11-tissue of liquid nitrogen.
Embodiment
Embodiment 1
In the present embodiment, the structure of aculeus type tissue slice glassivation freezing carrier is made up of frozen pipe 1 and needling device as shown in Figure 1.Frozen pipe 1 is a pipe, and end sealing, an end opening are made length 40mm, internal diameter 6mm of PE (polyethylene) material.The structure of needling device such as Fig. 8, shown in Figure 9, by syringe needle 7 and the pin 8 that is used to wear tissue form, the lower section of syringe needle 7 is coniform with the interior shape of frozen pipe 1 opening end and size match, its upper segment is rectangular plate shape (retaining part of vitrifying cooling operation), pin 8 is two, make of stainless steel, its diameter 0.14mm, length 30mm, phase is installed in the lower end of syringe needle 7 across a certain distance.The array mode of frozen pipe 1 and needling device as shown in Figure 1, after the combination, pin 8 inserts in the frozen pipe 1 from the opening end of frozen pipe 1, syringe needle 7 be the pipe close of frozen pipe 1, and the opening end of frozen pipe 1 is sealed.
In the present embodiment, the structure of aculeus type tissue slice glassivation freezing carrier is made up of frozen pipe 1 and needling device as shown in Figure 2.Frozen pipe 1 is a pipe, the sealing of one end, an end opening, make of PE (polyethylene) material, length 50mm, internal diameter 6mm is provided with dividing plate 2 in the frozen pipe, spacing L1 between described dividing plate and the blind end is 10mm, and the spacing L2 between the opening end is 40mm, and decompression filler cotton cord 4 is equipped with in the space that dividing plate 2, blind end and the tube wall between them surround, and the closed end wall of dividing plate 2, frozen pipe 1 has the vent 3 that the aperture is 1.5mm.The structure of needling device as shown in Figure 3, by long handle 5, short handle 6, syringe needle 7 and the pin 8 that is used to wear tissue form, long handle 5 and short handle 6 are integrated, long handle 5 is the contraction structure that is easy to fracture with the boundary of short handle 6, and the lower section of syringe needle 7 be coniform with the interior shape of frozen pipe 1 opening end and size match, and its upper end and short handle 6 are connected, pin 8 is one, make of stainless steel, its diameter 0.18mm, length 35mm are installed in the lower end of syringe needle 7.The array mode of frozen pipe 1 and needling device as shown in Figure 2, after the combination, pin 8 inserts in the frozen pipe 1 from the opening end of frozen pipe 1, syringe needle 7 be the pipe close of frozen pipe 1, and the opening end of frozen pipe 1 is sealed.
In the present embodiment, the aculeus type tissue slice glassivation freezing carrier is made up of frozen pipe 1 and needling device.Frozen pipe 1 is identical with embodiment 1; The structure of needling device as shown in Figure 4, by long handle 5, short handle 6, syringe needle 7 and the pin 8 that is used to wear tissue form, long handle 5 and short handle 6 are two members, the lower section of long handle 5 is a cone, the upper segment of short handle 6 is provided with the conical bore that is complementary with long handle 5, and long handle 5 is connected for plug-in with short handle 6.The lower section of syringe needle 7 is coniform with the interior shape of frozen pipe 1 opening end and size match, and its upper end is connected with short handle 6, and pin 8 is one, makes its diameter 0.16mm, length 35mm of stainless steel.After the combination, pin 8 inserts in the frozen pipe 1 from the opening end of frozen pipe 1, and syringe needle 7 is the pipe close of frozen pipe 1, with the opening end sealing of frozen pipe 1.
In the present embodiment, the aculeus type tissue slice glassivation freezing carrier is made up of frozen pipe 1 and needling device.The shape of frozen pipe 1 is identical with embodiment 2 with the architecture form, and difference from Example 2 is: frozen pipe 1 length 60mm internal diameter 12mm, and the spacing L1 between dividing plate and the blind end is 10mm, and the spacing L2 between the opening end is 50mm; The structure of needling device such as Fig. 5, shown in Figure 6, difference from Example 2 is that the pin 8 that is used to wear tissue is three, length 42mm is the lower end that " triangle " shape is installed in syringe needle 7 mutually across a certain distance.Three pins also can be " word " shape across a certain distance and be installed in the lower end of syringe needle 7, as shown in Figure 7.
In the present embodiment, the aculeus type tissue slice glassivation freezing carrier is made up of frozen pipe 1 and needling device.The shape of frozen pipe 1 is identical with embodiment 2 with the architecture form, and difference from Example 2 is: the internal diameter 12mm of frozen pipe 1; The structure of needling device such as Figure 10, shown in Figure 11, by syringe needle 7 and the pin 8 that is used to wear tissue form, the lower section of syringe needle 7 is coniform with the interior shape of frozen pipe 1 opening end and size match, its upper segment is rectangular plate shape (retaining part of vitrifying cooling operation), pin 8 is four, make of stainless steel, its diameter 0.12mm, length 35mm are the lower end that " rectangle " shape is installed in syringe needle 7 mutually across a certain distance.。
Present embodiment uses embodiment 2 described aculeus type tissue slice glassivation freezing carriers to carry out glass freezing to freezing ovary tissue, operating method as shown in figure 12, operation steps is as follows:
(1) the people's ovary cortex sheet that obtains of will performing the operation is divided into area 1～4mm in the culture dish that fills specimen collection liquid (the Leibovitz-15 substratum adds 10% foetal calf serum or albumin or synthetic blood serum substituting composition)
2The ovary tissue sheet, 2～5mm spacing of then 4-6 sheet ovary tissue sheet being separated by is through on the stainless steel needle 8;
(2) hand-held long handle 5 stainless steel needle 8 that will be installed with the ovary tissue sheet was transferred in inferior maple and 7.5% (v/v) ethylene glycol of first kind of cryoprotectant 7.5% (v/v) dimethyl in inferior maple and 15% (v/v) ethylene glycol of dehydration 10 minutes, second kind cryoprotectant 15% (v/v) dimethyl dehydration 2 minutes successively, inhale removal ovary with antiseptic gauze then and organize the cryoprotectant on sheet surface, the stainless steel needle 8 that will be installed with the ovary tissue sheet is again put into the container 9 that fills liquid nitrogen;
(3) with long handle tweezer 10 frozen pipe 1 is tilted to put into the container 9 that fills liquid nitrogen, make liquid nitrogen enter frozen pipe 1, hand-held then long handle 5 will be installed with the ovary tissue sheet in liquid nitrogen stainless steel needle 8 is put into frozen pipe 1, and syringe needle 7 is as the opening end sealing of pipe close with frozen pipe 1;
(4) behind the cracking-off long handle 5, the freezing carrier that liquid nitrogen and ovary tissue sheet are housed of step 3 assembling is transferred to liquid nitrogen container stores.
Claims (9)
1, a kind of aculeus type tissue slice glassivation freezing carrier, the frozen pipe (1) that comprises end sealing, an end opening, it is characterized in that also comprising needling device, described needling device comprises syringe needle (7) and is installed in the pin (8) that is used to wear tissue on the syringe needle that syringe needle (7) is provided with and the interior shape of frozen pipe (1) opening end and portion's section of size match; After needling device and frozen pipe (1) combination, pin (8) is positioned at frozen pipe (1), and syringe needle (7) is that the pipe close of frozen pipe (1) seals frozen tube opening end.
2, aculeus type tissue slice glassivation freezing carrier according to claim 1, it is characterized in that being provided with in the described frozen pipe (1) dividing plate (2), decompression filler (4) is equipped with in the space that this dividing plate and blind end and the tube wall between them surround, and the closed end wall of dividing plate (2), frozen pipe (1) is or/and have vent (3) on the tube wall between them.
3, aculeus type tissue slice glassivation freezing carrier according to claim 2, the spacing L1 that it is characterized in that dividing plate (2) and frozen pipe (1) blind end are dividing plates (2) and 1/5th to 1/4th of the spacing L2 of frozen pipe (1) opening end; Decompression filler (4) is cotton cord or cotton yarn.
4, according to the described aculeus type tissue slice glassivation freezing carrier of arbitrary claim in the claim 1 to 3, the pin (8) that it is characterized in that being used to wear tissue is at least one, and its diameter is not more than 0.2mm.
5, aculeus type tissue slice glassivation freezing carrier according to claim 4 is characterized in that needling device also comprises handle, and described handle is connected with syringe needle (7).
6, aculeus type tissue slice glassivation freezing carrier according to claim 5, it is characterized in that described handle is made up of long handle (5) and short handle (6), long handle (5) and short handle (6) are integrated, and long handle (5) is the contraction structure that is easy to fracture with the boundary of short handle (6).
7, aculeus type tissue slice glassivation freezing carrier according to claim 5 is characterized in that described handle is made up of long handle (5) and short handle (6), and long handle (5) is connected for plug-in with short handle (6).
8, a kind of method of glass freezing ovary tissue is characterized in that freezing carrier is the described arbitrary aculeus type tissue slice glassivation freezing carrier of claim 1 to 5, and operation steps is as follows:
(1) the people's ovary cortex sheet that obtains of will performing the operation is divided into the ovary tissue sheet in the culture dish that fills specimen collection liquid, is through at least 4 ovary tissue sheets on the pin (8) then with being separated with spacing mutually;
(2) pin (8) that will be installed with the ovary tissue sheet is transferred in the cryoprotectant after the dehydration, organizes the cryoprotectant on sheet surface with sterilization water-absorbing material suction removal ovary, and the pin (8) that will be installed with the ovary tissue sheet is again put into the container (9) that fills liquid nitrogen;
(3) frozen pipe (1) is put into the container (9) that fills liquid nitrogen, and made the liquid nitrogen frozen pipe (1) of packing into, the pin (8) that will be installed with the ovary tissue sheet then in liquid nitrogen is put into frozen pipe (1), and syringe needle (7) is as the opening end sealing of pipe close with frozen pipe (1);
(4) freezing carrier that liquid nitrogen and ovary tissue sheet are housed of step (3) assembling being transferred to liquid nitrogen container stores.
9, a kind of method of glass freezing ovary tissue is characterized in that freezing carrier is claim 6 or 7 described aculeus type tissue slice glassivation freezing carriers, and operation steps is as follows:
(1) the people's ovary cortex sheet that obtains of will performing the operation is divided into the ovary tissue sheet in the culture dish that fills specimen collection liquid, is through at least 4 ovary tissue sheets on the pin (8) then with being separated with spacing mutually;
(2) hand-held long handle (5) pin (8) that will be installed with the ovary tissue sheet is transferred in the cryoprotectant after the dehydration, inhale removal ovary with the sterilization water-absorbing material and organize the cryoprotectant on sheet surface, the pin (8) that will be installed with the ovary tissue sheet is again put into the container (9) that fills liquid nitrogen;
(3) frozen pipe (1) is put into the container (9) that fills liquid nitrogen, and make the liquid nitrogen frozen pipe (1) of packing into, hand-held then long handle (5) will be installed with the ovary tissue sheet in liquid nitrogen pin (8) is put into frozen pipe (1), and syringe needle (7) is as the opening end sealing of pipe close with frozen pipe (1);
(4) remove long handle (5) after, the freezing carrier that liquid nitrogen and ovary tissue sheet are housed of step (3) assembling is transferred to liquid nitrogen container stores.
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| CN200810045568XA CN101314757B (en) | 2008-07-15 | 2008-07-15 | Aculeus type tissue slice glassivation refrigeration carrier and refrigeration method for ovary tissue |
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| CN200810045568XA CN101314757B (en) | 2008-07-15 | 2008-07-15 | Aculeus type tissue slice glassivation refrigeration carrier and refrigeration method for ovary tissue |
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