CN1572870A - Method for storing human ovocyte - Google Patents
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- CN1572870A CN1572870A CN 200410042875 CN200410042875A CN1572870A CN 1572870 A CN1572870 A CN 1572870A CN 200410042875 CN200410042875 CN 200410042875 CN 200410042875 A CN200410042875 A CN 200410042875A CN 1572870 A CN1572870 A CN 1572870A
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Abstract
A method for storing human ovocyte is characterized in that: firstly, placing the human ovocyte into a cryoprotective liquor containing sucrose and 1,2-propylene glycol (PROH) to make pretreatment, then making programmed slowly cooling and freezing, then storing it into liquid nitrogen. When it is used, quickly heating and reanimating the human ovocyte, and placing it into sequential culture solution and placing it into cultivated box with 37 DEG C plus or minus 0.2 DEG C and 5%-6% CO<2> concentration to make culture for making external fertilization. The method can improve the survival ratio for the cooled human ovocyte, is simple in operation, and requires simple, reasonable price, low cost, and high reanimate rate instruments and solution, and is in favour of solving the human infertility problem and the carry out and execution of the birth control national policy.
Description
Technical field
The invention belongs to the field of biomedicine technology of reproductive medicine, be a kind of storage procedures of human oocyte, exactly, relate to a kind of storage human oocyte method that adopts slow cooling cryogenic freezing and quick-thawing recovery.
Background technology
The low tempertaure storage of human oocyte is extremely important to the treatment of infertility.At first, because at vitro fertilization-embryo implanting (IVF-EF, in vitro fertilization and embryo transfer) raising of short super ovulation technology and embryo culture technology in the technology, feasible (the IVF in vitro fertilization of half nearly, in vitrofertilization) treatment cycle has remaining ovocyte, if can this part human oocyte is frozen, after recovery, put back to uterine cavity again, can increase the chance of becoming pregnant.Secondly, human oocyte frozen has and need not in time to supply sperm, also can be applicable to also avoid ethics problem simultaneously to giving advantages such as treatment because of birth defect or because of the infertile patient of putting, chemotherapy, operative treatment lose ovarian function implements ovum.In addition, than embryo cryopreservation more superiority is arranged from the frozen of angle human oocyte of preserving Fertility, especially for because of the patient of putting, chemotherapy, operative treatment might lose ovarian function, the frozen of its ovocyte is the sole mode that keeps Fertility.
Though the human sperm is freezing very ripe with embryo cryopreservation, conventional already applying clinical, human ovocyte freeze thawing problem is not resolved always.Its major cause is: (1) human oocyte is the cell of human body maximum, and its cytoplasm is very abundant, surface-area and volume ratio are little, is difficult for fully dehydration; (2) membrane permeability of human oocyte is poor, is not easy dehydration; (3) because human oocyte is in the reduction division MII phase, the relatively poor and cytoskeleton (comprising spindle body etc.) of its membrane stability is to freezing responsive especially, and causes the low and chromosome abnormalty of recovery back survival rate, causes fertilization and grows failure; (4) to form the recovery of every functions such as embryo also very difficult in the human oocyte that is in the reduction division MII phase its reduction division and fertilization after freeze thawing.
Therefore, how to improve the survival rate of human oocyte after frozen, and the recovery of the every function assessment in freezing recovery back is present insider's problem demanding prompt solution.People's reported first such as chen were made the frozen human oocyte of cryoprotectant with DMSO (dimethyl sulfoxide (DMSO)) in 1986, obtained 76% survival rate.The research that people such as Porcu in 1997 carry out large sample obtains 54% survival rate, and with the freeze thawing ovum through the ICSI fertilization childbirth that secures good health.Set up the U.S. first ovum in medical college of the geographic University of Wisconsin of Chicago,U.S in July, 2002, the method that adopts is the supper-fast freeze-thaw method of vitrifying, though its anabiosis rate is 93.5%, but its method implements greatly but effect instability of difficulty, now existing 3 example gestation, 335 ovums of stock.In addition, domestic minority scholar has reported their the preliminary experiment result of study about the egg freezing technology, its anabiosis rate is 73.66%, rate of fertilization is 75%, the embryo quality rate of formation is 30.6%, 6-8 cell stage rate of formation is 12.24%, and draws the conclusion that the granulosa cell that wraps quilt helps egg freezing.Mostly be at present the research of small sample amount in the world about the report of egg freezing, perhaps the big effect instability of difficulty perhaps fluctuates at 50%-90% more than the anabiosis rate, also not can be used for routine clinical stable method.In a word, it is very undesirable that existing Refrigeration Technique is carried out the effect of frozen human oocyte, and anabiosis rate is low and very unstable.
Summary of the invention
The purpose of this invention is to provide a kind of storage procedures of human oocyte, this method can improve survival rate and reduction division, the fertilization recovery that form every functions such as embryo thereof of human oocyte after frozen, and is easy and simple to handle, expense is low; Solved the problem that effect is undesirable, anabiosis rate is low of the frozen human oocyte of existing Refrigeration Technique preferably.
The object of the present invention is achieved like this: a kind of storage procedures of human oocyte, earlier human oocyte is placed on and contains sucrose (Sucrose) and 1, carry out freezing pre-treatment in the frozen solution of 2 propylene glycol (PROH), the slow cooling of carrying out sequencing again is freezing, carries out low tempertaure storage then in liquid nitrogen; When needs use, this human oocyte is rapidly heated thaw and makes its recovery; This human oocyte of recovery of will thawing is again put into sequential culture liquid, inserts the back of recovering in 37 ± 0.2 ℃, the incubator of 5%～6% gas concentration lwevel and cultivates, in vitro fertilization; Described method comprises following concrete operations step:
A, freezing pre-treatment: get after the ovum,, remove ovarian cumulus tissue around the human oocyte, this human oocyte is placed on carries out freezing treatment in the frozen solution more earlier through 2-3 hour hatch;
B, programmed cooling is freezing and low tempertaure storage: pretreated human oocyte is packed in the straw, put into the program frigorimeter, slowly cooling is freezing to carry out sequencing; When temperature reaches the low-temperature values of setting, and after stable 10-20 minute, this human oocyte is moved into low tempertaure storage in the liquid nitrogen, standby for recovery;
C, be rapidly heated and thaw: from liquid nitrogen, take out and store the straw that this human oocyte is arranged, successively be placed in the air at room temperature and in 30 ± 3 ℃ the water, make it rapid intensification; Again straw is dried, cut off, take out this human oocyte, sequentially put into the different recovery treatment solutions that thaws again and recover;
Cultivate D, recovery back: this human oocyte of the recovery of will thawing is put into sequential culture liquid, inserts 37 ± 0.2 ℃, 5%～6%CO
2Cultivate in the concentration incubator, in vitro fertilization.
Above-mentioned frozen solution includes A liquid, B liquid and three kinds of liquid of C liquid;
Described A liquid (volume ratio) is that its serum or SSS volumn concentration are 15～25% with the solution of phosphate buffered saline buffer (PBS, phosphate buffer) or HTF-HEPES uterine tube liquid damping fluid dilute serum or SSS gained;
Described B liquid is to be the PROH liquid of element task liquid preparation with A liquid, and its PROH concentration is 1.0～2.0M;
Described C liquid is to be the mixed solution that contains PROH and Sucrose of element task liquid preparation with A liquid, and its PROH concentration is 1.0～2.0M, and Sucrose concentration is 0.25～0.35M.
Described freezing pre-treatment further comprises following operation steps:
A1, get after the ovum, through 2～3 hours hatch, the method that combines with enzyme and machinery was removed ovarian cumulus tissue around the human oocyte, puts into incubator cultivation 0.5～1 hour again;
A2, B liquid and two kinds of solution of C liquid are placed under the room temperature balance respectively take out respectively and place vessel in right amount after 1～2 hour;
A3, from incubator, take out human oocyte, place B liquid to soak 5～15 minutes this human oocyte earlier after, in C liquid, soaked 10～20 minutes again, begin the set up procedure frigorimeter this moment.
Further comprise following operation steps among the described step B:
B1, begin in C liquid the human oocyte straw of packing into, the tubulature method is: inhale the C liquid 2.5 ± 1.0cm → air 1.5 ± 1.0cm → C liquid 1.5 ± 1.0cm of C liquid 1.5 ± 1.0cm → air 1.5 ± 1.0cm → contain human oocyte → seal with straw;
B2, the straw that human oocyte will be housed are put into frigorimeter, and to carry out programmed cooling freezing, its cooling process is: from+15～25 ℃, when first rate of temperature fall with-2 ± 1.0 ℃/minute drops to-7 ± 0.1 ℃, ice is planted at the place at no human oocyte fluid column, again when-7 ± 0.1 ℃ begins to continue slowly to cool to-30 ± 5 ℃ with-0.3 ± 0.1 ℃/minute rate of temperature fall, again with-30 ± 5 ℃/minute rate of temperature fall fast cooling to-175 ± 5 ℃, after stablizing 10-20 minute, the straw that again this is equipped with human oocyte moves into low tempertaure storage in-196 ℃ of liquid nitrogen rapidly.
The described recovery treatment solution that thaws includes above-mentioned A liquid and D liquid, E liquid, F liquid;
Described D liquid is to be the PROH liquid of element task liquid preparation with A liquid, and its PROH concentration is 0.5～1.5M, and Sucrose concentration is 0.25～0.35M;
Described E liquid is to be the mixed solution that contains PROH and Sucrose of element task liquid preparation with A liquid, and its PROH concentration is 0.2～1.2M, and Sucrose concentration is 0.25～0.35M;
Described F liquid is to be the Sucrose liquid that element task liquid is prepared with A liquid, and its Sucrose concentration is 0.25～0.35M.
Further comprise the following operation steps that is rapidly heated and thaws among the described step C:
C1, be that the strainer sequencing of 0.22 μ m filters F liquid, E liquid and D liquid with the sieve aperture specification, for standby;
C2, freezing straw is taken out from liquid nitrogen, place air after 30～50 seconds, place 30 ± 3 ℃ of water to soak again 30～50 seconds;
C3, straw is dried, cut off, wherein human oocyte was successively placed D liquid and E liquid respectively each 2～10 minutes; Again it is successively placed F liquid and A liquid respectively each 5～15 minutes, then this human oocyte was moved in 37 ± 0.2 ℃ of incubators in the equilibrated A liquid balance 5～15 minutes.
The time of cultivating in the incubator among the described step D is 2 to 2.5 hours, after cultivating in vitro fertilization.
The storage procedures of human oocyte of the present invention has scientific rationality, and success ratio is higher.At first, in the preprocessing process of this method, owing to adopt the method that the ovarian cumulus tissue (promptly wrapping the granulosa cell of quilt) around the human oocyte is removed, help the survival after the freezing recovery of human oocyte, and can make later fertilization divide the potentiality that develop into the embryo to be not fully exerted.The reservation of its major cause granulosa cell affects in the human oocyte refrigerating process rehydration in dehydration and the recovery process.Referring to table.
Whether granulosa cell around table one ovocyte removes the influence to the freeze thawing result
| Group | The A group | The B group | The C group |
| Survival rate | ????93％ | ????96％ | ????6％ |
| Rate of fertilization | ????82％ | ????80％ | ????0 |
| The embryo quality rate of formation | ????89％ | ????90％ | ????0 |
From table 1 obviously as can be known, remove granulosa cell more fully or keep 2～3 layers of granulosa cell and do not remove granulosa cell fully diverse result is arranged, and the survival rate of the human oocyte of removing granulosa cell more fully and keeping 2～3 layers of granulosa cell after freezing reaches 93% and 96% respectively, and its rate of fertilization and embryo quality rate can reach 82%, 80% and 89%, 90% respectively.Frozen solution in the method; as C liquid; its sucrose concentration adopts 0.25M～0.35M; especially be best with 0.3M; more help human oocyte thaw the recovery after form and functional rehabilitation, thereby make human oocyte survival rate, high-quality ovum pick-up rate, rate of fertilization and embryo quality rate of formation reach higher and stable (referring to table two).
Different sucrose concentration is to human oocyte freeze thawing result's influence in table two frozen solution
| Group | A organizes (∠ 0.25M) | B group (0.25～0.35M) |
| Total survival rate | ????∠30％ | ????91％ |
| High-quality ovum pick-up rate | ????∠24％ | ????90％ |
| Rate of fertilization | ????∠69％ | ????81％ |
| The embryo quality rate of formation | ????∠13％ | ????89％ |
Show that from the experimental result of table two concentration is too low or too high, and recovery after freezing all has bigger influence (not providing in the too high experimental data table of relevant concentration) to human oocyte.In addition; the time that exposes in frozen solution before human oocyte is freezing is selected science for use; soaked in C liquid 10～20 minutes in 5～15 minutes as soaking in B liquid earlier, the inside and outside osmotic pressure of favourable cell reaches balance and fully dehydration again, can prevent the toxic action of frozen solution pair cell.Thereby the survival rate of favourable raising human oocyte and rate of fertilization afterwards and embryo quality rate of formation (referring to table three).
Starting time before table three ovocyte is freezing in refrigerating fulid is to freeze thawing result's influence
Because exposure duration is long, with the effect of pair cell toxigenicity, is unfavorable for the survival of cell.The experiment proved that exposure duration is long, the survival rate of cell will reduce.Secondly, recovery treatment solution in the recovery of thawing, sucrose concentration adopts 0.25M～0.35M, is best especially with 0.3M, also helps the recovery of refrigerated human oocyte.Moreover, in recovery was cultivated, the incubation time after its recovery was controlled at 2～2.5 hours, was fertilized afterwards again, help the potentiality recovery that freezing recovery descendant Oocyte Meiosis and fertilization division later on develop into the embryo, thereby improved the rate of formation (referring to table four) of embryo quality.
| Group | A organizes (∠ 15min) | B group (15～35min) |
| Survival rate | ?????∠58％ | ????91％ |
| Rate of fertilization | ?????∠69％ | ????81％ |
| The embryo quality rate of formation | ?????∠25％ | ????89％ |
The different incubation times in table four recovery back are to human oocyte freeze thawing result's influence
| Group | The A group (＜1.5h) | B group (1.5～2.5h) | The C group (＞2.5h) |
| Total survival rate | ??????91％ | ????91％ | ????91％ |
| High-quality ovum pick-up rate | ??????89％ | ????89％ | ????89％ |
| Rate of fertilization | ????∠54％ | ????63％ | ????81％ |
| The embryo quality rate of formation | ????∠47％ | ????60％ | ????89％ |
In a word, the present invention is that a kind of human oocyte is adopted frozen the storage procedures of melting soon slowly.Survival rate by the human oocyte of this freezing method for resuscitation after freezing recovery can be stabilized in about 90%, the rate of fertilization 82% of recovery ovum behind ICSI, fertilization embryo division rate 93%, the rate of formation 89% of embryo quality, 6-8 cell stage rate of formation 87%, karyomit(e) nuclearity type analysis is not found the aneuploid phenomenon.This human oocyte freeze-thaw technology has reached international most advanced level, and clinical application effect significantly its clinical pregnancy rate reaches 47%, is similar to the success ratio of the fresh cycles with embryo transfers of the tube baby treatment cycle of egg freezing (promptly without).This method not only improve human oocyte after frozen survival rate and recovery after be fertilized, divide, develop into embryo's potentiality, and it is easy and simple to handle, required medical apparatus and solution are simple, inexpensive, and cost is low, help solving carrying out of the human infertile problem and the national policy of family planning.
Description of drawings
Fig. 1 is the schematic flow sheet of the inventive method.
Embodiment
Referring to Fig. 1, the present invention is a kind of storage procedures of human oocyte, this method is human oocyte to be placed on to contain sucrose (Sucrose) and 1 earlier, soak freezing pre-treatment 1 in the frozen solution of 2 propylene glycol (PROH), the slow cooling of carrying out sequencing again is freezing, low tempertaure storage is promptly lowered the temperature freezing and low tempertaure storage 2 in liquid nitrogen then; When needs use, this human oocyte is rapidly heated thaws 3, make it recovery, this human oocyte of the recovery of will thawing is again put into sequential culture liquid, inserts 37 ± 0.2 ℃, 5%～6% carbonic acid gas CO
2Cultivate 4 in the incubator of concentration, in vitro fertilization.
Storage procedures below in conjunction with the concrete introducer's ovocyte of embodiments of the invention.
The concrete operations step of first embodiment is described earlier:
At first, above-mentioned B liquid and C liquid being placed under the room temperature balance respectively took out 1.5ml respectively and places sterile vessel after 1.5 hours; From incubator, take out human oocyte then, place above-mentioned B liquid immersion after 15 minutes the ovocyte of having removed the ovarian cumulus tissue, in C liquid, soaked 12 minutes again; While start-up routine frigorimeter.Above-mentioned A liquid, B liquid and C liquid can be prepared in the following way: (select the human body uterine tube liquid damping fluid HTF-HEPES of 42ml for use currently available products, be common articles for use in the medical experiment, (also select currently available products for use with the human serum of 7.4ml down together), be the common articles for use in the medical experiment, down with) to be hybridly prepared into the serum volumn concentration be 15% solution, i.e. A liquid; Then the PROH liquid that A liquid and the 1.0ml concentration of 8.5ml is 9.5M is hybridly prepared into the PROH solution of 1.0M, i.e. B liquid; Adding 1.0ml concentration at the A of 8.5ml liquid again is that PROH liquid and the 0.975gSucrose (molecular weight is 342) of 9.5M is hybridly prepared into the mixed solution that contains 1.0MPROH and 0.3Msucrose, i.e. C liquid; Use strainer (its sieve aperture specification is 0.22 micron) sequencing to filter B liquid and C liquid again, get final product.At last, surplus solution can be put into 4 ℃ of refrigerators, and preservation period is a week.
Carry out the programmed cooling freezing treatment then: take out this human oocyte straw of packing into when being dipped into 12 minutes in C liquid, concrete tubulature method is: inhale the C liquid 3.0cm → air 2.0cm → C liquid 2.5cm of C liquid 2.5cm → air 2.0cm → wherein contain human oocyte → seal with straw.At this moment, the straw that human oocyte is housed is put into frigorimeter, and to carry out programmed cooling freezing, its cooling process is: from+18 ℃, when first rate of temperature fall with-1.5 ℃/minute drops to-7 ℃, after keeping 2 minutes, plant ice (seeding) in the place of no human oocyte fluid column, after keeping 10 minutes again, again when-7 ℃ begin to continue to cool to-30 ℃ lentamente with-0.3 ℃/minute rate of temperature fall, again with-30 ℃/minute rate of temperature fall fast cooling to-172 ℃ of low temperature, after stablizing 10 minutes, this straw that human oocyte is housed is moved into liquid nitrogen (196 ℃) low tempertaure storage.
Before taking out the human oocyte of low tempertaure storage, at first should measure required effective D liquid, E liquid and F liquid carrying out in vitro fertilization.About D liquid, E liquid and F liquid can be prepared in the following manner: the A liquid 9.2ml for preparing with aforesaid way also adds PROH liquid and the 0.855g Sucrose that 0.8ml concentration is 10M and is mixedly configured into the PROH of 0.8M and the Sucrose mixed solution of 0.25M, i.e. D liquid; Again with the 9.6mlA liquid of aforesaid way preparation and add PROH liquid and the 0.855gSucrose that 0.4ml concentration is 10M and be mixedly configured into PROH and the Sucrose mixed solution of 0.25M, i.e. the E liquid that contains 0.4M; Also 9mlA liquid and the 0.855g Sucrose with preparation by the way is hybridly prepared into the solution that contains 0.25MSucrose, i.e. F liquid; Be 0.22 micron strainer sequencing filtration F liquid, E liquid and D liquid at last with sieve aperture, for standby.
The step of recovering of thawing is: earlier freezing straw is taken out from liquid nitrogen, place air after 35 seconds, place 30 ℃ of water to soak again 45 seconds; Again straw is dried, cut off, wherein human oocyte was placed on earlier in D liquid and the E liquid each 5 minutes; Again it successively was positioned over respectively in F liquid and the A liquid each 15 minutes, and human oocyte was moved in 37 ℃ of incubators, placed 15 minutes in the equilibrated A liquid then, at last it is moved in sequential culture liquid, at 37 ℃, 5%CO
2Cultivate in the incubator of concentration after 2 hours, take out and carry out monosperm microinjection fertilization in the ooecium slurry.
The concrete operations step of another embodiment of the present invention is:
At first, above-mentioned B liquid and C liquid being placed under the room temperature balance respectively took out 1.2ml respectively and places sterile vessel after 2 hours; From incubator, take out human oocyte then, place above-mentioned B liquid immersion after 7 minutes the ovocyte of having removed ovarian cumulus, in C liquid, soaked 20 minutes again; While start-up routine frigorimeter.Above-mentioned A liquid, B liquid and C liquid can be made in the following manner: it is 15% solution that the human serum of the human body uterine tube liquid damping fluid HTF-HEPES of 42ml and 14ml is hybridly prepared into the serum volumn concentration, i.e. A liquid; Then the PROH liquid that A liquid and the 2.0ml concentration of 8.0ml is 10M is hybridly prepared into the solution of 2.0MPROH, i.e. B liquid; The A liquid of getting 8.0ml again adds PROH liquid and the 1.197g Sucrose that 2.0ml concentration is 10M therein and is hybridly prepared into the mixed solution that contains 2.0MPROH and 0.35MSucrose, i.e. C liquid; Use strainer (its sieve aperture specification is 0.22 micron) sequencing to filter B liquid and C liquid again, for standby.
Carry out the programmed cooling freezing treatment then: take out this human oocyte straw of packing into when being dipped into 20 minutes in C liquid, concrete tubulature method is: inhale the C liquid 2.5cm → air 2.5cm → C liquid 2.0cm of C liquid 2.0cm → air 2.5cm → wherein contain human oocyte → seal with straw.At this moment, the straw that human oocyte is housed is put into frigorimeter, and to carry out programmed cooling freezing, its cooling process is: from+24 ℃, when first rate of temperature fall with-2.5 ℃/minute drops to-7 ℃, after keeping 3 minutes, plant ice (seeding) in the place of no human oocyte fluid column, after keeping 10 minutes again, again when-7 ℃ begin to continue to cool to-35 ℃ lentamente with-0.4 ℃/minute rate of temperature fall, again with-35 ℃/minute rate of temperature fall fast cooling to-180 ℃ of low temperature, after stablizing 15 minutes, this straw that human oocyte is housed is moved into low tempertaure storage in the liquid nitrogen (196 ℃).
Carry out at needs at first should measuring required effective D liquid, E liquid and F liquid before the human oocyte of in vitro fertilization and taking-up low tempertaure storage.About A liquid, D liquid, E liquid and F liquid can be prepared in the following manner: earlier being hybridly prepared into the serum volumn concentration with the human serum of the HTF-HEPES human body uterine tube liquid damping fluid of 42ml and 14ml is 25% solution, i.e. A liquid; Then, the A liquid of 8.5ml being added 1.5ml concentration is that PROH liquid and the 1.197g Sucrose of 10M is mixedly configured into PROH and the Sucrose mixed solution of 0.35M, i.e. the D liquid that contains 1.5M; The PROH liquid and the 1.197gSucrose that the A liquid of 9ml are added 1ml concentration again and be 10M are mixedly configured into PROH and the Sucrose mixed solution of 0.35M, i.e. the E liquid that contains 1.0M; Also A liquid and the 1.197g Sucrose with 9ml is hybridly prepared into the solution that contains 0.35MSucrose, i.e. F liquid; Be 0.22 micron strainer sequencing filtration F liquid, E liquid and D liquid at last with sieve aperture, for standby.
The step of recovering of thawing is: earlier freezing straw is taken out from liquid nitrogen, place air at room temperature after 45 seconds, place 32 ℃ of water to soak again 35 seconds; Again straw is dried, cut off, wherein human oocyte was placed on earlier in D liquid and the E liquid each 8 minutes; Again it successively was positioned over respectively in F liquid and the A liquid each 8 minutes, and human oocyte was moved in 37 ℃ of incubators, placed 10 minutes in the equilibrated A liquid then, at last it is moved in sequential culture liquid, at 37 ℃, 5%CO
2Cultivate in the incubator of concentration after 2.5 hours, take out and carry out monosperm microinjection fertilization in the ooecium slurry.
In the foregoing description, in the process for preparation of B liquid, C liquid and D liquid, E liquid, the PROH concentration that is adopted is inconsistent, just in order to illustrate that its compound method can adopt different technique means.
Used mechanical means is existing general-purpose equipment among the present invention, no longer illustrates at this.
The present invention has tested enforcement repeatedly, and the result of test is successful, has realized goal of the invention.
Claims (7)
1, a kind of storage procedures of human oocyte, earlier human oocyte is placed on and contains sucrose (Sucrose) and 1, carry out freezing pre-treatment in the frozen solution of 2 propylene glycol (PROH), the slow cooling of carrying out sequencing again is freezing, carries out low tempertaure storage then in liquid nitrogen; When needs use, this human oocyte is rapidly heated thaw and makes its recovery; This human oocyte of recovery of will thawing is again put into sequential culture liquid, inserts the back of recovering in 37 ± 0.2 ℃, the incubator of 5%～6% gas concentration lwevel and cultivates, in vitro fertilization; It is characterized in that described method comprises following concrete operations step:
A, freezing pre-treatment: get after the ovum,, remove ovarian cumulus tissue around the human oocyte, this human oocyte is placed on carries out freezing treatment in the frozen solution more earlier through 2-3 hour hatch;
B, programmed cooling is freezing and low tempertaure storage: pretreated human oocyte is packed in the straw, put into the program frigorimeter, slowly cooling is freezing to carry out sequencing; When temperature reaches the low-temperature values of setting, and after stable 10-20 minute, this human oocyte is moved into low tempertaure storage in the liquid nitrogen, standby for recovery;
C, be rapidly heated and thaw: from liquid nitrogen, take out and store the straw that this human oocyte is arranged, successively be placed in the air at room temperature and in 30 ± 3 ℃ the water, make it rapid intensification; Again straw is dried, cut off, take out this human oocyte, sequentially put into the different recovery treatment solutions that thaws again and recover;
Cultivate D, recovery back: this human oocyte of the recovery of will thawing is put into sequential culture liquid, inserts 37 ± 0.2 ℃, 5%～6%CO
2Cultivate in the concentration incubator, in vitro fertilization.
2, the storage procedures of human oocyte according to claim 1 is characterized in that: described frozen solution includes A liquid, B liquid and three kinds of liquid of C liquid;
Described A liquid (volume ratio) is that its serum or SSS volumn concentration are 15～25% with the solution of phosphate buffered saline buffer (PBS, phosphate buffer) or HTF-HEPES uterine tube liquid damping fluid dilute serum or SSS gained;
Described B liquid is to be the PROH liquid of element task liquid preparation with A liquid, and its PROH concentration is 1.0～2.0M;
Described C liquid is to be the mixed solution that contains PROH and Sucrose of element task liquid preparation with A liquid, and its PROH concentration is 1.0～2.0M, and Sucrose concentration is 0.25～0.35M.
3, the storage procedures of human oocyte according to claim 2 is characterized in that: described freezing pre-treatment further comprises following operation steps:
A1, get after the ovum, through 2-3 hour hatch, the method that combines with enzyme and machinery was removed ovarian cumulus tissue around the human oocyte, puts into incubator cultivation 0.5-1 hour again;
A2, B liquid and two kinds of solution of C liquid are placed under the room temperature balance respectively take out respectively and place vessel in right amount after 1～2 hour;
A3, from incubator, take out human oocyte, place B liquid to soak 5～15 minutes this human oocyte earlier after, in C liquid, soaked 10～20 minutes again, begin the set up procedure frigorimeter this moment.
4, according to the storage procedures of claims 3 described human oocytes, it is characterized in that: further comprise following operation steps among the described step B:
B1, begin in C liquid the human oocyte straw of packing into, the tubulature method is: inhale the C liquid 2.5 ± 1.0cm → air 1.5 ± 1.0cm → C liquid 1.5 ± 1.0cm of C liquid 1.5 ± 1.0cm → air 1.5 ± 1.0cm → contain human oocyte → seal with straw;
B2, the straw that human oocyte will be housed are put into frigorimeter, and to carry out programmed cooling freezing, its cooling process is: from+15～25 ℃, when first rate of temperature fall with-2 ± 1.0 ℃/minute drops to-7 ± 0.1 ℃, ice is planted at the place at no human oocyte fluid column, again when-7 ± 0.1 ℃ begins to continue slowly to cool to-30 ± 5 ℃ with-0.3 ± 0.1 ℃/minute rate of temperature fall, again with-30 ± 5 ℃/minute rate of temperature fall fast cooling to-175 ± 5 ℃, after stablizing 10-20 minute, the straw that again this is equipped with human oocyte moves into low tempertaure storage in-196 ℃ of liquid nitrogen rapidly.
5, according to the storage procedures of claims 4 described human oocytes, it is characterized in that: the described recovery treatment solution that thaws includes above-mentioned A liquid and D liquid, E liquid, F liquid;
Described D liquid is to be the PROH liquid of element task liquid preparation with A liquid, and its PROH concentration is 0.5～1.5M, and Sucrose concentration is 0.25～0.35M;
Described E liquid is to be the mixed solution that contains PROH and Sucrose of element task liquid preparation with A liquid, and its PROH concentration is 0.2～1.2M, and Sucrose concentration is 0.25～0.35M;
Described F liquid is to be the Sucrose liquid that element task liquid is prepared with A liquid, and its Sucrose concentration is 0.25～0.35M.
6, according to the storage procedures of claims 5 described human oocytes, it is characterized in that: further comprise the following operation steps that is rapidly heated and thaws among the described step C:
C1, be that the strainer sequencing of 0.22 μ m filters F liquid, E liquid and D liquid with the sieve aperture specification, for standby;
C2, freezing straw is taken out from liquid nitrogen, place air after 30～50 seconds, place 30 ± 3 ℃ of water to soak again 30～50 seconds;
C3, straw is dried, cut off, wherein human oocyte was successively placed D liquid and E liquid respectively each 2～10 minutes; Again it was successively placed F liquid and A liquid respectively each 5～15 minutes, then this human oocyte was moved in 37 ± 0.2 ℃ of incubators in the equilibrated A liquid 5～15 minutes.
7, according to the storage procedures of claim 1 or 6 described human oocytes, it is characterized in that: the time of cultivating in the incubator among the described step D is 2 to 2.5 hours, after cultivating in vitro fertilization.
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| CNB2004100428754A CN100482785C (en) | 2003-05-30 | 2004-05-28 | Method for storing human ovocyte |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100582221C (en) * | 2008-09-04 | 2010-01-20 | 昆明理工大学 | Ultra low temperature freezing-storage method for onosma paniculatum cell |
| CN104663649A (en) * | 2015-02-13 | 2015-06-03 | 安徽医科大学 | Human ovocyte cryoprotectant |
| CN106857498A (en) * | 2016-12-27 | 2017-06-20 | 科兴（大连）疫苗技术有限公司 | A kind of cell cryopreservation protection liquid and its application without dimethyl sulfoxide (DMSO) |
| CN108841717A (en) * | 2018-05-31 | 2018-11-20 | 陈诗梦 | A kind of thawing apparatus freezing egg cell |
-
2004
- 2004-05-28 CN CNB2004100428754A patent/CN100482785C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100582221C (en) * | 2008-09-04 | 2010-01-20 | 昆明理工大学 | Ultra low temperature freezing-storage method for onosma paniculatum cell |
| CN104663649A (en) * | 2015-02-13 | 2015-06-03 | 安徽医科大学 | Human ovocyte cryoprotectant |
| CN106857498A (en) * | 2016-12-27 | 2017-06-20 | 科兴（大连）疫苗技术有限公司 | A kind of cell cryopreservation protection liquid and its application without dimethyl sulfoxide (DMSO) |
| CN106857498B (en) * | 2016-12-27 | 2021-05-14 | 科兴（大连）疫苗技术有限公司 | Cell cryopreservation protective solution without dimethyl sulfoxide and application thereof |
| CN108841717A (en) * | 2018-05-31 | 2018-11-20 | 陈诗梦 | A kind of thawing apparatus freezing egg cell |
| CN108841717B (en) * | 2018-05-31 | 2021-06-29 | 张艳 | Thawing apparatus for frozen egg cells |
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| CN100482785C (en) | 2009-04-29 |
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