JP2021016336A - Method for cryopreserving mammalian early embryo - Google Patents
Method for cryopreserving mammalian early embryo Download PDFInfo
- Publication number
- JP2021016336A JP2021016336A JP2019133275A JP2019133275A JP2021016336A JP 2021016336 A JP2021016336 A JP 2021016336A JP 2019133275 A JP2019133275 A JP 2019133275A JP 2019133275 A JP2019133275 A JP 2019133275A JP 2021016336 A JP2021016336 A JP 2021016336A
- Authority
- JP
- Japan
- Prior art keywords
- embryo
- embryos
- solution
- thawing
- early
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 210000001161 mammalian embryo Anatomy 0.000 title claims abstract description 96
- 238000000034 method Methods 0.000 title claims abstract description 69
- 241000283973 Oryctolagus cuniculus Species 0.000 claims abstract description 34
- 238000002844 melting Methods 0.000 claims abstract description 32
- 230000008018 melting Effects 0.000 claims abstract description 32
- 239000007788 liquid Substances 0.000 claims abstract description 30
- 230000004083 survival effect Effects 0.000 claims abstract description 18
- 238000010257 thawing Methods 0.000 claims description 54
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 23
- 229930006000 Sucrose Natural products 0.000 claims description 23
- 239000005720 sucrose Substances 0.000 claims description 23
- 238000005138 cryopreservation Methods 0.000 claims description 15
- 241000124008 Mammalia Species 0.000 claims description 7
- 238000004017 vitrification Methods 0.000 abstract description 28
- 238000003776 cleavage reaction Methods 0.000 abstract description 16
- 230000007017 scission Effects 0.000 abstract description 16
- 241001465754 Metazoa Species 0.000 abstract description 3
- 230000006866 deterioration Effects 0.000 abstract 1
- 210000002257 embryonic structure Anatomy 0.000 description 80
- 239000000243 solution Substances 0.000 description 67
- 241000700159 Rattus Species 0.000 description 33
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 32
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 27
- 210000004027 cell Anatomy 0.000 description 17
- 229910052757 nitrogen Inorganic materials 0.000 description 16
- 238000007710 freezing Methods 0.000 description 12
- 230000008014 freezing Effects 0.000 description 12
- 230000018109 developmental process Effects 0.000 description 11
- 230000003204 osmotic effect Effects 0.000 description 11
- 210000002459 blastocyst Anatomy 0.000 description 8
- 235000013601 eggs Nutrition 0.000 description 8
- 239000012530 fluid Substances 0.000 description 8
- 230000035899 viability Effects 0.000 description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 239000013078 crystal Substances 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 230000013020 embryo development Effects 0.000 description 5
- 229920001917 Ficoll Polymers 0.000 description 4
- 210000001124 body fluid Anatomy 0.000 description 4
- 239000010839 body fluid Substances 0.000 description 4
- 239000002577 cryoprotective agent Substances 0.000 description 4
- 238000004321 preservation Methods 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 210000003722 extracellular fluid Anatomy 0.000 description 3
- 239000000644 isotonic solution Substances 0.000 description 3
- 239000003761 preservation solution Substances 0.000 description 3
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 2
- 229920001342 Bakelite® Polymers 0.000 description 2
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 108010003272 Hyaluronate lyase Proteins 0.000 description 2
- 102000001974 Hyaluronidases Human genes 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 2
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 2
- 210000004102 animal cell Anatomy 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 210000001771 cumulus cell Anatomy 0.000 description 2
- 230000002542 deteriorative effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 229960002773 hyaluronidase Drugs 0.000 description 2
- 239000000815 hypotonic solution Substances 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000027758 ovulation cycle Effects 0.000 description 2
- 230000010412 perfusion Effects 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000010902 straw Substances 0.000 description 2
- 239000003656 tris buffered saline Substances 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 239000006145 Eagle's minimal essential medium Substances 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 241000186660 Lactobacillus Species 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- NPYPAHLBTDXSSS-UHFFFAOYSA-N Potassium ion Chemical compound [K+] NPYPAHLBTDXSSS-UHFFFAOYSA-N 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- FKNQFGJONOIPTF-UHFFFAOYSA-N Sodium cation Chemical compound [Na+] FKNQFGJONOIPTF-UHFFFAOYSA-N 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 239000006096 absorbing agent Substances 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000007798 antifreeze agent Substances 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 229910001424 calcium ion Inorganic materials 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 230000004720 fertilization Effects 0.000 description 1
- 239000000819 hypertonic solution Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 208000000509 infertility Diseases 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 231100000535 infertility Toxicity 0.000 description 1
- 230000009027 insemination Effects 0.000 description 1
- 239000007951 isotonicity adjuster Substances 0.000 description 1
- 229940039696 lactobacillus Drugs 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000007758 minimum essential medium Substances 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 229910001414 potassium ion Inorganic materials 0.000 description 1
- OJTDGPLHRSZIAV-UHFFFAOYSA-N propane-1,2-diol Chemical compound CC(O)CO.CC(O)CO OJTDGPLHRSZIAV-UHFFFAOYSA-N 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 229910001415 sodium ion Inorganic materials 0.000 description 1
- 229960002920 sorbitol Drugs 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
本発明は、凍結保存後の哺乳動物初期胚を融解する方法や、凍結保存された哺乳動物初期胚を含む胚移植用キットに関する。 The present invention relates to a method for thawing an early mammalian embryo after cryopreservation, and an embryo transfer kit containing the cryopreserved early mammalian embryo.
哺乳動物において、雌の生殖器から回収した受精卵（初期胚）や体外受精によって作製した胚を、レシピエント（受卵動物）に移植する胚移植技術は広く普及している。この技術において、胚は、レシピエントの性周期に応じた時期に移植する必要があるため、生存性や機能を低下させずに胚を凍結保存する技術が必要とされる。現在までに、胚の凍結保存として、緩慢法（非特許文献１）、ガラス化法（非特許文献２）、超急速ガラス化法（非特許文献３）等が知られている。 In mammals, embryo transfer techniques for transplanting fertilized eggs (early embryos) collected from female reproductive organs or embryos produced by in vitro fertilization to recipients (recipients) are widely used. In this technique, embryos need to be transplanted at a time corresponding to the recipient's sexual cycle, so a technique for cryopreserving embryos without deteriorating viability or function is required. To date, a slow method (Non-Patent Document 1), a vitrification method (Non-Patent Document 2), an ultra-rapid vitrification method (Non-Patent Document 3), and the like are known as cryopreservation of embryos.
ガラス化法とは、グリセロールやエチレングリコール、ＤＭＳＯ（ジメチルスルホキシド）などの耐凍剤を多量に含む水溶液の凝固点降下により、氷点下でも氷晶が形成されにくくなる原理を用いたものである。この水溶液を急速に液体窒素中で冷却させると氷晶が形成されないまま固体化させることができる。また、超急速ガラス化法とは、胚を少量のガラス化液とともに液体窒素に浸漬するという方法である。ガラス化保存法を用いると、細胞内外の何れにも氷晶が生じないために凍結時及び融解時の細胞への物理的障害（凍害）を回避することができるが、ガラス化液に含まれる耐凍剤には化学的毒性があり、細胞の凍結保存時にはガラス化液が少ない方が好ましく、さらには解凍後ただちにガラス化液を希釈する必要がある。 The vitrification method uses the principle that ice crystals are less likely to be formed even below freezing point due to the freezing point depression of an aqueous solution containing a large amount of freezing agent such as glycerol, ethylene glycol, and DMSO (dimethyl sulfoxide). When this aqueous solution is rapidly cooled in liquid nitrogen, it can be solidified without forming ice crystals. The ultra-rapid vitrification method is a method in which an embryo is immersed in liquid nitrogen together with a small amount of vitrifying solution. When the vitrification preservation method is used, it is possible to avoid physical damage (freezing damage) to the cells at the time of freezing and thawing because ice crystals do not occur inside or outside the cells, but it is contained in the vitrified solution. The antifreeze agent is chemically toxic, and it is preferable that the amount of the vitrification solution is small when the cells are cryopreserved, and it is necessary to dilute the vitrification solution immediately after thawing.
これらガラス化保存法を用いた卵子、胚の凍結保存法としては様々なものが提案されている。例えば、特許文献１及び２には、ストローにガラス化液を充満させた中で卵子又は胚をガラス化保存させ、解凍時に素早く希釈液と接触させて再生率を向上させる試みがなされている。また、特許文献３には、卵子又は胚の周囲に付着した余分なガラス化液を濾紙などの吸収体により吸収させることにより、優れた生存性で凍結保存させる方法が開示されている。さらに、特許文献４及び５では、人の不妊治療分野で使用されているクライオトップ法という方法で、卵付着保持用ストリップとして短冊状の可撓性かつ無色透明なフィルムを使用し、該フィルム上にごく少量のガラス化液と共に卵子又は胚を顕微鏡下で付着させ、凍結保存する方法が開示されている。 Various cryopreservation methods for eggs and embryos using these vitrification preservation methods have been proposed. For example, Patent Documents 1 and 2 attempt to vitrify and store an egg or embryo in a straw filled with a vitrifying solution and quickly bring it into contact with a diluting solution at the time of thawing to improve the regeneration rate. Further, Patent Document 3 discloses a method of cryopreserving with excellent survivability by absorbing an excess vitrified solution adhering to the periphery of an egg or an embryo with an absorber such as filter paper. Further, in Patent Documents 4 and 5, a strip-shaped flexible and colorless transparent film is used as a strip for holding egg adhesion by a method called a cryotop method used in the field of human infertility treatment, and the film is used. A method is disclosed in which an egg or embryo is attached under a microscope together with a very small amount of vitrification solution and cryopreserved.
しかし、クライオトップ法は一度に１０個以下の少数の胚しか凍結保存できないこと、また、使用する器具が高額であるという問題点があり、ラット等の実験動物で使用することは実用的ではない。このため、胚の生存率や機能を維持しながら、より大量の胚を安価で凍結保存するための技術の開発が求められていた。 However, the cryotop method has problems that only a small number of embryos of 10 or less can be cryopreserved at a time and that the equipment used is expensive, so that it is not practical to use it in experimental animals such as rats. .. Therefore, there has been a demand for the development of a technique for inexpensively cryopreserving a larger amount of embryos while maintaining the survival rate and function of embryos.
本発明の課題は、クライオチューブ内で凍結保存された哺乳動物初期胚を、その生存率や質（例えば、胚発生能）を低下させることなく融解する方法を提供することにある。 An object of the present invention is to provide a method for thawing an early mammalian embryo cryopreserved in a cryotube without deteriorating its viability or quality (eg, embryonic development potential).
本発明者らは、以前より上記課題を解決すべく鋭意研究を重ね、凍結保存された哺乳動物初期胚の融解速度が、融解後の胚の生存率及び質に大きな影響を及ぼす可能性を示唆してきた（Seki S and Mazur P, Cryobiology and Cryotechnology， 62, No. 2, 105-108, 2016、Seki S et al.Cryobiology, 81, 132-137, 2018）。しかし、当該技術分野においては、初期胚を含む動物細胞は、タンパク質の変性等を防ぐために生体内での温度（３７〜３９度程度）以下の条件下で操作しなければならないと信じられており、上記文献においても、本発明者らは、融解速度を高めるために２３℃又は３７℃の溶液を使用するに留まっていた。 The present inventors have conducted intensive studies to solve the above problems, and suggested that the thawing rate of cryopreserved early mammalian embryos may have a great influence on the survival rate and quality of embryos after thawing. (Seki S and Mazur P, Cryobiology and Cryotechnology, 62, No. 2, 105-108, 2016, Seki S et al. Cryobiology, 81, 132-137, 2018). However, in the art, it is believed that animal cells, including early embryos, must be manipulated under conditions below in vivo temperature (about 37-39 degrees) to prevent protein denaturation. Also in the above literature, the present inventors have only used a solution at 23 ° C. or 37 ° C. to increase the melting rate.
一方、本研究において、本発明者らは凍結保存されたラット及びウサギの１細胞期胚を、５０℃という高温の融解用液を用いて急速融解することより、いずれの動物種においても融解後の生存率、卵割率、及び／又は発生率が向上するという予想外の実験結果を得た。本発明の融解工程においては、ラット及びウサギの１細胞期胚は一時的に５０℃近い高温になるが、それでも、急速融解により胚の質が維持されたことは、当業者にとって驚くべきことである。本発明は、以上のような予想外の知見に基づいて、はじめて完成されたものである。 On the other hand, in this study, the present inventors rapidly thawed cryopreserved 1-cell stage embryos of rats and rabbits using a thawing solution at a high temperature of 50 ° C., after thawing in all animal species. Unexpected experimental results were obtained that the survival rate, cleavage rate, and / or incidence rate of the cells were improved. In the thawing process of the present invention, rat and rabbit 1-cell stage embryos temporarily reach a high temperature of nearly 50 ° C., but it is surprising to those skilled in the art that the quality of the embryos was maintained by rapid thawing. is there. The present invention has been completed for the first time based on the above unexpected findings.
すなわち、本発明は、（１）凍結状態の哺乳動物初期胚を４０〜６０℃の融解用液と接触させる融解工程を含む、凍結保存後の前記哺乳動物初期胚を融解する方法や、（２）融解用液が５０℃である、上記（１）に記載の方法や、（３）融解用液がスクロースを含む溶液である、上記（１）又は（２）に記載の方法や、（４）哺乳動物初期胚が、ガラス化されたものである、上記（１）〜（３）のいずれかに記載の方法や、（５）哺乳動物初期胚が、クライオチューブ内で凍結保存されている、上記（１）〜（４）のいずれかに記載の方法や、（６）哺乳動物が、ラット又はウサギである、上記（１）〜（５）のいずれかに記載の方法や、（７）初期胚が、１細胞期胚である、上記（１）〜（６）のいずれかに記載の方法や、（８）凍結保存後の哺乳動物初期胚の生存率及び／又は発生率を改善するための、上記（１）〜（７）のいずれかに記載の方法に関する。 That is, the present invention includes (1) a method of thawing the early mammalian embryo after cryopreservation, which comprises a thawing step of bringing the frozen early mammalian embryo into contact with a thawing solution at 40 to 60 ° C., and (2). ) The method according to (1) above, wherein the melting solution is 50 ° C., (3) the method according to (1) or (2) above, where the melting solution is a solution containing a mammal, or (4). ) The method according to any one of (1) to (3) above, wherein the early mammalian embryo is vitrified, and (5) the early mammalian embryo is cryopreserved in a cryotube. , The method according to any one of (1) to (4) above, (6) the method according to any one of (1) to (5) above, wherein the mammal is a rat or a rabbit, or (7). ) The method according to any one of (1) to (6) above, wherein the early embryo is a 1-cell stage embryo, and (8) improving the survival rate and / or development rate of the early mammalian embryo after cryopreservation. The method according to any one of (1) to (7) above.
また、本発明は、（９）凍結状態の哺乳動物初期胚を４０〜６０℃の融解用液と接触させる融解工程を行う旨が記載された説明書が共にパッケージされた、凍結保存された哺乳動物初期胚を含む胚移植用キットや、（１０）５０℃の融解用液と接触させる融解工程を行う旨が記載された説明書が共にパッケージされた、上記（９）に記載のキットや、（１１）スクロースを含む融解用液をさらに備える、上記（９）又は（１０）に記載のキットや、（１２）哺乳動物初期胚が、ガラス化されたものである、上記（９）〜（１１）のいずれかに記載のキットや、（１３）哺乳動物初期胚が、クライオチューブ内で凍結保存されている、上記（９）〜（１２）のいずれかに記載のキットや、（１４）哺乳動物が、ラット又はウサギである、上記（９）〜（１３）のいずれかに記載のキットや、（１５）初期胚が、１細胞期胚である、上記（９）〜（１４）のいずれかに記載のキットに関する。 In addition, the present invention is a cryopreserved mammal in which (9) a description stating that a thawing step of bringing a frozen early mammalian embryo into contact with a thawing solution at 40 to 60 ° C. is performed together is packaged. The kit according to (9) above, which is packaged together with a kit for embryo transplantation containing early animal embryos and (10) instructions for performing a melting step in contact with a melting solution at 50 ° C. (11) The kit according to (9) or (10) above, further comprising a thawing solution containing sucrose, and (12) early mammalian embryos, which are vitrified. The kit according to any one of 11), the kit according to any one of (9) to (12) above, in which the early mammalian embryo is cryopreserved in a cryotube, or (14). The kit according to any one of (9) to (13) above, wherein the mammal is a rat or a rabbit, and (15) the early embryo is a one-cell stage embryo, according to (9) to (14) above. For any of the kits listed.
本発明によれば、クライオトップよりも安価なクライオチューブを用いて、哺乳動物初期胚を大量に凍結保存することが可能となる。 According to the present invention, it is possible to cryopreserve a large amount of early mammalian embryos by using a cryotube which is cheaper than a cryotop.
本発明の「凍結保存後の哺乳動物初期胚を融解する方法」としては、凍結状態の哺乳動物初期胚を４０〜６０℃の融解用液と接触させる融解工程を含むものであれば特に制限されない（以下、かかる方法を、「本発明の方法」と称する場合がある）。上記本発明の方法は、凍結保存後の哺乳動物初期胚の生存率及び／又は発生率を改善するために用いることができ、ここで「生存率及び／又は発生率を改善する」とは、融解工程において生じる哺乳動物初期胚への物理的又は機能的な損傷を軽減し、融解後の初期胚における（ｉ）生存率、（ii）卵割率、（iii）胚盤胞期までの発生率、又は（iv）受胎率の低下を抑制若しくは防止することを意味する。上記「哺乳動物初期胚」としては、ラット、ウサギ、マウス、ウシ、ブタ、ハムスター、イヌ、サル、ヤギ、ネコ、ヒツジ等の非ヒト哺乳動物由来の初期胚、及び／又はヒト由来の初期胚であれば特に制限されないが、非ヒト哺乳動物由来の初期胚であることが好ましく、ラット又はウサギ由来の初期胚であることがさらに好ましい。また、上記「初期胚」としては、１細胞期胚から胚盤胞段階までの発生初期段階の胚であればどのようなものであってもよいが、１細胞期胚（通常、胚発生開始後１６時間以内）、２細胞期胚（通常、胚発生開始後１６〜２４時間の範囲内）、４細胞期胚（通常、胚発生開始後約４８時間）、又は８細胞期胚（通常、胚発生開始後約７２時間）であることが好ましく、１細胞期胚であることが特に好ましい（以下、かかる哺乳動物初期胚を単に「胚」と称する場合がある）。 The "method for thawing early mammalian embryos after cryopreservation" of the present invention is not particularly limited as long as it includes a thawing step in which the early mammalian embryos in a frozen state are brought into contact with a thawing solution at 40 to 60 ° C. (Hereinafter, such a method may be referred to as "the method of the present invention"). The method of the present invention can be used to improve the survival rate and / or development rate of early mammalian embryos after cryopreservation, where "improving survival rate and / or development rate" means It reduces the physical or functional damage to early mammalian embryos that occurs during the thawing process, and (i) viability, (ii) cleavage rate, and (iii) development up to the blastocyst stage in the early embryos after thawing. It means suppressing or preventing a decrease in rate, or (iv) conception rate. The "mammalian early embryos" include early embryos derived from non-human mammals such as rats, rabbits, mice, cows, pigs, hamsters, dogs, monkeys, goats, cats, and sheep, and / or human-derived early embryos. However, the embryo is not particularly limited, but is preferably an early embryo derived from a non-human mammal, and more preferably an early embryo derived from a rat or rabbit. The "early embryo" may be any embryo in the early stage of development from the 1-cell stage embryo to the embryonic stage, but the 1-cell stage embryo (usually, embryonic development start). 2 cell stage embryos (usually within 16 to 24 hours after embryonic development), 4 cell stage embryos (usually about 48 hours after embryonic development), or 8 cell stage embryos (usually within 16 hours) It is preferably about 72 hours after the start of embryogenesis), and particularly preferably a 1-cell stage embryo (hereinafter, such an early mammalian embryo may be simply referred to as an “embryo”).
本発明の方法に用いる「胚」は、緩慢法、ガラス化法、超急速ガラス化法などの公知の方法で凍結保存されたものであれば特に制限されない。ここで、「緩慢法」とは、比較的低濃度の凍結保護剤を含む保存液中で、胚を０．３〜０．５℃／分の速度で−３０℃前後まで冷却した後に、液体窒素に浸漬して凍結する方法であり、大がかりな装置を用いることなく凍結保存後の胚を融解することが可能であるが、冷却過程において、細胞外に氷晶が形成され、この氷晶が胚に物理的な損傷を与えるため、胚の生存性及び受胎率が低くなる傾向があることが知られている。一方、「ガラス化法」とは、高濃度の凍結保護剤を含む保存液（ガラス化液）を用いて、胚を液体窒素に浸漬して液体窒素の温度まで急速に冷却する方法であり、また、「超急速ガラス化法」とは、胚を少量のガラス化液と共に液体窒素に浸漬する方法であり、これらの方法では、細胞内外での氷晶の形成を抑制して、細胞の内外をガラス化する方法であり、緩慢法と比較して胚の生存性及び受胎率を高く保つことができる。 The "embryo" used in the method of the present invention is not particularly limited as long as it is cryopreserved by a known method such as a slow method, a vitrification method, or an ultra-rapid vitrification method. Here, the "slow method" is a liquid after cooling the embryo to about -30 ° C at a rate of 0.3 to 0.5 ° C / min in a storage solution containing a relatively low concentration cryoprotectant. It is a method of immersing in nitrogen and freezing, and it is possible to thaw embryos after cryopreservation without using a large-scale device, but in the cooling process, ice crystals are formed outside the cells, and these ice crystals are formed. It is known that embryo viability and conception rate tend to decrease due to physical damage to the embryo. On the other hand, the "vitrification method" is a method in which an embryo is immersed in liquid nitrogen and rapidly cooled to the temperature of liquid nitrogen using a preservation solution (vitrification solution) containing a high concentration of cryoprotectant. The "ultra-rapid vitrification method" is a method of immersing an embryo in liquid nitrogen together with a small amount of vitrifying solution. In these methods, the formation of ice crystals inside and outside the cell is suppressed, and the inside and outside of the cell are suppressed. It is a method of vitrifying the liquid, and can maintain high embryo viability and conception rate as compared with the slow method.
したがって、本発明の方法に用いる「胚」は、ガラス化法又は超急速ガラス化法により凍結保存された胚、すなわち、細胞内に凍結保護剤が浸透するように処理された、ガラス化された胚であることが特に好ましい。上記「ガラス化液」としては、例えば、エチレングリコール、フィコール、スクロース、グリセロール、ＤＭＳＯ（ジメチルスルホキシド）、プロピレングリコール（１，２−プロパンジオール）等の凍結保護剤を単独又は組み合わせて含有する溶液であれば特に制限されないが、エチレングリコール、フィコール、及びスクロースを組み合わせて含有するＥＦＳ溶液を特に好適に挙げることができる。また、上記「ガラス化された胚」は、上記ガラス化液に浸漬又は懸濁された状態で、クライオチューブ（低温チューブ）、クライオトップ、クライオストロー（低温ストロー）、クライオバッグ（低温バッグ）等の凍結保存用容器中に保存されていることが好ましく、クライオチューブ又はクライオトップ中に保存されていることがより好ましく、クライオチューブ中に保存されていることが特に好ましい。 Therefore, the "embryo" used in the method of the present invention is an embryo that has been cryopreserved by a vitrification method or an ultra-rapid vitrification method, that is, a vitrified embryo that has been treated so that the cryoprotectant penetrates into the cell. It is particularly preferred to be an embryo. The above-mentioned "vitrification solution" is, for example, a solution containing a cryoprotectant such as ethylene glycol, Ficoll, sucrose, glycerol, DMSO (dimethyl sulfoxide), and propylene glycol (1,2-propanediol) alone or in combination. If there is no particular limitation, an EFS solution containing a combination of ethylene glycol, Ficoll, and sucrose can be particularly preferably mentioned. Further, the above-mentioned "vitrified embryo" is immersed in or suspended in the above-mentioned vitrification solution, and has a cryotube (low temperature tube), a cryotop, a cryostraw (low temperature straw), a cryobag (low temperature bag), etc. It is preferably stored in a cryopreservation container, more preferably stored in a cryotube or a cryotop, and particularly preferably stored in a cryotube.
本発明の方法における「融解工程」は、上記凍結保存方法によって凍結状態にある胚を、４０〜６０℃の範囲内に保持された融解用液と接触させて、急速融解することを特徴とする。上記「融解用液」の温度としては、４０〜６０℃の範囲内であれば特に制限されないが、例えば、４２〜６０℃、４５〜６０℃、４７〜６０℃、４２〜５７℃、４５〜５７℃、４７〜５７℃、４２〜５５℃、４５〜５５℃、４７〜５５℃、４２〜５３℃、４５〜５３℃、４７〜５３℃、４８〜５２℃、４８〜５１℃、４９〜５１℃等の範囲であることが好ましく、４７、４８、４９、５０、５１、５２、又は５３℃であることがより好ましく、５０℃であることがさらに好ましい。また、上記融解工程における融解速度としては、例えば、６，８００℃／分以上であることが好ましく、７，２００℃／分以上であることがより好ましく、７，５００℃／分以上であることがさらに好ましく、７，８００℃／分以上であることが特に好ましい。 The "thawing step" in the method of the present invention is characterized in that embryos frozen by the above-mentioned cryopreservation method are brought into contact with a thawing solution held in the range of 40 to 60 ° C. and rapidly thawed. .. The temperature of the "melting liquid" is not particularly limited as long as it is within the range of 40 to 60 ° C., but for example, 42 to 60 ° C., 45 to 60 ° C., 47 to 60 ° C., 42 to 57 ° C., 45 to 57 ° C, 47-57 ° C, 42-55 ° C, 45-55 ° C, 47-55 ° C, 42-53 ° C, 45-53 ° C, 47-53 ° C, 48-52 ° C, 48-51 ° C, 49- The temperature is preferably in the range of 51 ° C., more preferably 47, 48, 49, 50, 51, 52, or 53 ° C., and even more preferably 50 ° C. The melting rate in the melting step is, for example, preferably 6,800 ° C./min or higher, more preferably 7,200 ° C./min or higher, and more preferably 7,500 ° C./min or higher. Is more preferable, and 7,800 ° C./min or higher is particularly preferable.
上記融解用液としては、「凍結状態の哺乳動物初期胚を融解するため」という用途に特定された液であれば特に制限されず、哺乳動物初期胚の生存が可能な液（例えば、等張液、低張液、高張液）であればよく、等張液を好適に例示することができる。本明細書において「等張液」とは、体液や細胞液の浸透圧とほぼ同じ浸透圧を有する液を意味し、具体的には、２５０〜３８０ｍＯｓｍ／Ｌの範囲内の浸透圧を有する液を意味する。また、本明細書において「低張液」とは、体液や細胞液の浸透圧よりも低い浸透圧を有する液を意味し、具体的には、２５０ｍＯｓｍ／Ｌ未満の浸透圧を有する液を意味する。かかる低張液としては、細胞が破裂しない程度の低張液（具体的には、１００〜２５０ｍＯｓｍ／Ｌ未満の範囲内の浸透圧を有する液）が好ましい。また、本明細書において「高張液」とは、体液や細胞液の浸透圧よりも高い浸透圧を有する液を意味し、具体的には、浸透圧が３８０ｍＯｓｍ／Ｌ超（好ましくは３８０ｍＯｓｍ／Ｌ超〜１０００ｍＯｓｍ／Ｌの範囲内）を意味する。 The thawing solution is not particularly limited as long as it is a solution specified for the purpose of "thawing a frozen early mammalian embryo", and a solution capable of surviving the early mammalian embryo (for example, isotonic). Any liquid, hypotonic liquid, hypertonic liquid) may be used, and an isotonic liquid can be preferably exemplified. As used herein, the term "isotonic fluid" means a fluid having an osmotic pressure substantially the same as the osmotic pressure of body fluid or extracellular fluid, and specifically, a fluid having an osmotic pressure in the range of 250 to 380 mOsm / L. Means. Further, in the present specification, the “hypotonic fluid” means a fluid having an osmotic pressure lower than the osmotic pressure of body fluid or extracellular fluid, and specifically, a fluid having an osmotic pressure of less than 250 mOsm / L. To do. As such a hypotonic solution, a hypotonic solution that does not cause cell rupture (specifically, a solution having an osmotic pressure within the range of 100 to 250 mOsm / L) is preferable. Further, in the present specification, the “hypertonic fluid” means a fluid having an osmotic pressure higher than the osmotic pressure of body fluid or extracellular fluid, and specifically, the osmotic pressure is more than 380 mOsm / L (preferably 380 mOsm / L). It means (within the range of super to 1000 mOsm / L).
上記等張液としては、体液や細胞液の浸透圧とほぼ同じになるようにナトリウムイオン、カリウムイオン、カルシウムイオン等によって塩濃度や糖濃度等を調整した等張液であれば特に制限されず、具体的には生理食塩水や、緩衝効果のある生理食塩水（例えば、ＰＢＳ、トリス緩衝生理食塩水［Tris BufferedSaline；ＴＢＳ］、ＨＥＰＥＳ緩衝生理食塩水）、リンゲル液、乳酸リンゲル液、酢酸リンゲル液、重炭酸リンゲル液、５％グルコース水溶液、動物細胞培養液（例えば、ＰＢ１、ＤＭＥＭ、ＥＭＥＭ、ＲＰＭＩ−１６４０、α−ＭＥＭ、Ｆ−１２、Ｆ−１０、Ｍ−１９９）、等張剤（例えば、ブドウ糖、Ｄ−ソルビトール、Ｄ−マンニトール、ラクトース、塩化ナトリウム）等を挙げることができ、後述する本実施例において、その効果が実証されているため、ＰＢ１が好ましい。等張液は、市販のものであっても、自ら調製したものであってもよい。 The isotonic solution is not particularly limited as long as it is an isotonic solution in which the salt concentration, sugar concentration, etc. are adjusted by sodium ion, potassium ion, calcium ion, etc. so as to be substantially the same as the osmotic pressure of body fluid or cell fluid. , Specifically, physiological saline, physiological saline having a buffering effect (for example, PBS, Tris Buffered Saline (TBS), HEPES buffered saline), Ringer's solution, Lactobacillus, Ringer's acetate, Heavy Ringer carbonate solution, 5% glucose aqueous solution, animal cell culture solution (for example, PB1, DMEM, EMEM, RPMI-1640, α-MEM, F-12, F-10, M-199), isotonic agent (for example, glucose, D-sorbitol, D-mannitol, lactose, sodium chloride) and the like can be mentioned, and PB1 is preferable because its effect has been demonstrated in this example described later. The isotonic solution may be a commercially available product or a self-prepared product.
上記融解用液としては、スクロース又はトレハロースを含むものが好ましく、かかるスクロースやトレハロースの濃度としては、例えば、０．２〜０．７Ｍの範囲内、好ましくは、約０．５Ｍ（０．４〜０．６Ｍの範囲内）である。 The melting liquid preferably contains sucrose or trehalose, and the concentration of such sucrose or trehalose is, for example, in the range of 0.2 to 0.7 M, preferably about 0.5 M (0.4 to 0.4 to M). Within the range of 0.6M).
上記融解工程において「胚を４０〜６０℃の融解用液と接触させる」方法としては、例えば、クライオチューブ内で凍結保存された胚を用いる場合には、該クライオチューブを液体窒素から取り出し、３０〜６０秒、好ましくは約６０秒間室温に置き、次に、上記胚を含む凍結液（例えば、ガラス化液）の１０〜３００倍量、好ましくは２００倍量の所定温度の融解用液を、上記クライオチューブに注入して胚を急速融解し、その後、３７℃以下の温度に保持した融解用液等で胚を洗浄すればよい。また、例えば、クライオトップ内で凍結保存された胚を用いる場合には、該クライオトップを液体窒素から取り出し、所定温度の融解用液中に直ちに浸漬することにより胚を急速融解し、その後、３７℃以下の温度に保持した融解用液等で胚を洗浄すればよい。かかる工程において、胚は一時的に４０〜６０℃という高温に曝されるが、それにも関わらず、融解後の胚の生存率や機能は高いまま維持される（以下の実施例を参照のこと）。 In the thawing step, as a method of "contacting the embryo with a thawing solution at 40 to 60 ° C.", for example, when using an embryo cryopreserved in a cryotube, the cryotube is taken out from liquid nitrogen and 30 Leave it at room temperature for ~ 60 seconds, preferably about 60 seconds, and then add 10 to 300 times, preferably 200 times, the amount of the freezing solution containing the embryo (for example, vitrification solution) for thawing at a predetermined temperature. The embryo may be rapidly thawed by injecting into the cryotube, and then the embryo may be washed with a melting solution or the like maintained at a temperature of 37 ° C. or lower. Further, for example, when using an embryo cryopreserved in a cryotop, the cryotop is taken out of liquid nitrogen and immediately immersed in a melting solution at a predetermined temperature to rapidly thaw the embryo, and then 37. The embryo may be washed with a melting solution or the like maintained at a temperature of ° C. or lower. In this step, the embryos are temporarily exposed to a high temperature of 40-60 ° C., but nevertheless, the viability and function of the embryos after thawing remain high (see Examples below). ).
さらに、本発明は、凍結保存された哺乳動物初期胚を含む胚移植用キットにも関する。かかるキットは、凍結状態の上記胚を４０〜６０℃の融解用液と接触させる融解工程を行う旨が記載された説明書が共にパッケージされたものであれば特に制限されないが、上記融解用液をさらに備えるものであることが好ましい。本発明のキットに含まれる胚はガラス化されたものであることが好ましく、また、かかる胚はクライオチューブ内で凍結保存されていることが特に好ましい。具体的には、本発明のキットとしては、１〜１００個、５０〜１００個、２５〜５０個、１０〜５０個、５〜２０個、７〜１５個、又は５〜１０個の胚、好ましくは、１００、７５、５０、３０、２０、１０、又は５個の胚が凍結保存されたクライオチューブを備えたものであって、かかる胚が３〜１００μＬ、５〜７５μＬ、５〜５０μＬ、５〜２５μＬ、又は１０〜２０μＬのガラス化液、好ましくは、３、５、１０μＬのガラス化液に懸濁された状態で凍結保存されているキットを好適に挙げることができる。また、本発明のキットに使用される融解用液は、上記クライオチューブとは別の容器に保存されていることが好ましく、その量は、上記ガラス化液の１０〜３００倍量であることが好ましく、２００倍量であることがより好ましい。 Furthermore, the present invention also relates to an embryo transfer kit containing cryopreserved early mammalian embryos. The kit is not particularly limited as long as it is packaged with an instruction manual stating that the frozen embryo is to be brought into contact with the thawing solution at 40 to 60 ° C., but the thawing solution is not particularly limited. It is preferable that the above is further provided. The embryos included in the kit of the present invention are preferably vitrified, and it is particularly preferable that such embryos are cryopreserved in a cryotube. Specifically, the kit of the present invention includes 1 to 100 embryos, 50 to 100 embryos, 25 to 50 embryos, 10 to 50 embryos, 5 to 20 embryos, 7 to 15 embryos, or 5 to 10 embryos. Preferably, 100, 75, 50, 30, 20, 10, or 5 embryos are provided with cryopreserved cryotubes, such embryos being 3-100 μL, 5-75 μL, 5-50 μL, Preferred examples include kits that are cryopreserved in a suspended state in 5 to 25 μL, or 10 to 20 μL of vitrification solution, preferably 3, 5, 10 μL of vitrification solution. Further, the melting liquid used in the kit of the present invention is preferably stored in a container separate from the cryotube, and the amount thereof is 10 to 300 times the amount of the vitrification liquid. It is preferable, and the amount is more preferably 200 times.
以下、実施例により本発明をより具体的に説明するが、本発明の技術的範囲はこれらの例示に限定されるものではない Hereinafter, the present invention will be described in more detail with reference to Examples, but the technical scope of the present invention is not limited to these examples.
（１）ラット１細胞期胚の凍結融解
（１−１）１細胞期胚の採取
発情期後期の成熟雌ラット（９−１６週齢）に、１５０ ＩＵ／ｋｇのＰＭＳＧを腹腔内投与し、さらに４８時間後に、７５ ＩＵ／ｋｇのｈＣＧを腹腔内投与した。その後、雄ラットと自然交配させ、ｈＣＧ投与から２４時間後に、０．１％のヒアルロニダーゼを含むＭ２培地を用いた卵管灌流により、受精卵（１細胞期胚）を回収した。回収後の１細胞期胚は、Ｍ２培地で洗浄した後に、０．５ｍｇ／ｍＬのヒアルロニダーゼを含むＭ２培地で懸濁することによって、卵丘細胞を除去した。以下、ここで得られたラット１細胞期胚を単に「ラット胚」と称する場合がある。
(1) Freezing and thawing of rat 1-cell stage embryos (1-1) Collection of 1-cell stage embryos 150 IU / kg of PMSG was intraperitoneally administered to mature female rats (9-16 weeks old) in the late estrous cycle. After an additional 48 hours, 75 IU / kg hCG was intraperitoneally administered. Then, it was spontaneously mated with a male rat, and 24 hours after the administration of hCG, a fertilized egg (1 cell stage embryo) was collected by oviductal perfusion using M2 medium containing 0.1% hyaluronidase. The recovered 1-cell stage embryos were washed with M2 medium and then suspended in M2 medium containing 0.5 mg / mL hyaluronidase to remove cumulus cells. Hereinafter, the rat 1-cell stage embryo obtained here may be simply referred to as "rat embryo".
（１−２）ラット胚のガラス化保存
図１に示す方法に従って、上記（１−１）で得られたラット胚をガラス化保存した。具体的には、上記ラット胚を、７．５％又は１０％のエチレングリコール（ＥＧ）を含む耐凍剤液中（３０μＬのドロップ）に浸漬して、２３℃で５〜１０分間インキュベートした。インキュベート後の胚を回収して、さらに、ガラス化保存液であるＥＦＳ４０溶液中［４０％（ｖ／ｖ）エチレングリコール、１８％（ｗ／ｖ）フィコール７０，及び０．３Ｍスクロースを含むＰＢ１液］（３０μＬのドロップ）中に浸漬した。そして、胚を含む５μＬのＥＦＳ４０溶液をクライオチューブ（凍結保存関連製品セラムチューブ、住友ベークライト株式会社製）に移してキャップをした。これらのラット胚とＥＦＳ４０溶液とを接触させる工程は、２３℃で合計１分となるように行った。キャップを閉めたクライオチューブを液体窒素に直接浸漬して、ガラス化したラット胚凍結し、以下の実験まで液体窒素中で保存した。
(1-2) Vitrification preservation of rat embryos The rat embryos obtained in (1-1) above were vitrified and preserved according to the method shown in FIG. Specifically, the rat embryos were immersed in a freezing agent solution (30 μL drop) containing 7.5% or 10% ethylene glycol (EG) and incubated at 23 ° C. for 5 to 10 minutes. Embryos after incubation are collected and further PB1 solution containing 40% (v / v) ethylene glycol, 18% (w / v) Ficoll 70, and 0.3M sucrose in EFS40 solution, which is a vitrification preservation solution. ] (30 μL drop). Then, 5 μL of EFS40 solution containing the embryo was transferred to a cryotube (cryopreservation-related product Serum tube, manufactured by Sumitomo Bakelite Co., Ltd.) and capped. The step of contacting these rat embryos with the EFS40 solution was carried out at 23 ° C. for a total of 1 minute. Cryotubes with closed caps were immersed directly in liquid nitrogen, vitrified rat embryos were frozen and stored in liquid nitrogen until the following experiments.
（１−３）ラット胚の融解
図２に示す方法に従って、上記（１−２）で凍結保存したラット胚を融解した。具体的には、０．５Ｍのスクロースを含むＰＢ１液（以下、「スクロース液」と称する場合がある）を調製して、２３℃、３７℃、及び５０℃の３つの温度のスクロース液を用意した。そして、上記クライオチューブを液体窒素から取り出してキャップを外し、チューブ内の液体窒素を捨てた後に、１ｍＬの上記スクロース液（２３℃、３７℃、又は５０℃）を各チューブに加えて、ラット胚を融解した。２３℃、３７℃、及び５０℃のスクロース液を用いた場合の融解速度、及び融解に要する時間（カッコ内に記載）はそれぞれ、４，６００℃／分（０．６秒）、６，６００℃／分（０．４秒）、７，８００℃／分（０．３秒）であった。融解したラット胚を、２３℃のスクロース液中（３０μＬのドロップ）に５分間浸した後に回収し、ＨＴＦ（１００μＬのドロップ）で培養した。１日間培養後のラット胚の生存率及び卵割率を測定した（ｎ＝３）。
(1-3) Thawing of rat embryos The rat embryos cryopreserved in (1-2) above were thawed according to the method shown in FIG. Specifically, a PB1 solution containing 0.5 M sucrose (hereinafter, may be referred to as “sucrose solution”) is prepared, and sucrose solutions having three temperatures of 23 ° C., 37 ° C., and 50 ° C. are prepared. did. Then, the cryotube is taken out from the liquid nitrogen, the cap is removed, the liquid nitrogen in the tube is discarded, and then 1 mL of the sucrose solution (23 ° C, 37 ° C, or 50 ° C) is added to each tube to add rat embryo. Was melted. The melting rate and the time required for melting (indicated in parentheses) when using sucrose solutions at 23 ° C, 37 ° C, and 50 ° C are 4,600 ° C / min (0.6 seconds) and 6,600, respectively. It was ° C./minute (0.4 seconds) and 7,800 ° C./min (0.3 seconds). The thawed rat embryos were soaked in a sucrose solution at 23 ° C. (30 μL drops) for 5 minutes, then collected and cultured in HTF (100 μL drops). The survival rate and cleavage rate of rat embryos after culturing for 1 day were measured (n = 3).
（１−４）ラット胚の生存率及び卵割率に及ぼす融解速度の影響
上記（１−３）により得られた結果を図３に示す。図３に示されるように、融解に要する時間が０．６秒であった群（融解温度が２３℃）では、ラット胚の生存率は２０％以下であり、卵割率はほぼ０％であった。また、融解に要する時間が０．４秒であった群（融解温度が３７℃）では、ラット胚の生存率は５０％程度まで上昇したが、卵割率は５％程度と低い水準であった。一方、融解に要する時間が０．３秒であった群（融解温度が５０℃）では、ラット胚の生存率及び卵割率はともに１００％に近く、無処理群（凍結融解をしていない新鮮ラット１細胞期胚）と同程度の高いレベルであった。
以上の結果から、融解速度が凍結ラット胚の生存性や機能に大きな影響を及ぼすことが明らかとなった。また、５０℃のスクロース液を用いて凍結ラット胚を急速融解（７，８００℃／分）することによって、新鮮胚と同等の高い生存率及び卵割率を維持できることが明らかとなった。
(1-4) Effect of melting rate on rat embryo survival rate and cleavage rate The results obtained by the above (1-3) are shown in FIG. As shown in FIG. 3, in the group in which the time required for thawing was 0.6 seconds (melting temperature was 23 ° C.), the survival rate of rat embryos was 20% or less, and the cleavage rate was almost 0%. there were. In the group in which the time required for thawing was 0.4 seconds (melting temperature was 37 ° C.), the survival rate of rat embryos increased to about 50%, but the cleavage rate was as low as about 5%. It was. On the other hand, in the group in which the time required for thawing was 0.3 seconds (thaw temperature was 50 ° C.), the survival rate and cleavage rate of rat embryos were both close to 100%, and the untreated group (not frozen and thawed). The level was as high as that of fresh rat 1-cell stage embryos.
From the above results, it was clarified that the thawing rate has a great influence on the viability and function of frozen rat embryos. Further, it was clarified that the high survival rate and cleavage rate equivalent to those of fresh embryos can be maintained by rapidly thawing (7,800 ° C./min) frozen rat embryos using a sucrose solution at 50 ° C.
（２）ウサギ１細胞期胚の凍結融解
（２−１）１細胞期胚の採取
発情期後期の成熟雌ウサギ（１６−２０週齢）に、１５０ ＩＵ／ｋｇのＰＭＳＧを腹腔内投与し、さらに７２時間後に、１００ ＩＵ／ｋｇのｈＣＧを腹腔内投与した。その後、採取したウサギ精子を用いて人工授精を施し、ｈＣＧ投与１６時間後に、１０％ＦＢＳを含む１９９培地（ＦＢＳ−１９９）を用いた卵管灌流により、受精卵（１細胞期胚）を回収した。回収後の１細胞期胚は、上記１９９培地で洗浄した後に、懸濁することによって、卵丘細胞を除去した。以下、ここで得られたウサギ１細胞期胚を単に「ウサギ胚」と称する場合がある。
(2) Freezing and thawing of rabbit 1-cell stage embryos (2-1) Collection of 1-cell stage embryos 150 IU / kg of PMSG was intraperitoneally administered to mature female rabbits (16-20 weeks old) in the late estrous cycle. After an additional 72 hours, 100 IU / kg hCG was intraperitoneally administered. Then, artificial insemination was performed using the collected rabbit sperm, and fertilized eggs (1 cell stage embryos) were collected by oviductal perfusion using 199 medium (FBS-199) containing 10% FBS 16 hours after hCG administration. did. The 1-cell stage embryos after recovery were washed with the above-mentioned 199 medium and then suspended to remove cumulus cells. Hereinafter, the rabbit 1-cell stage embryo obtained here may be simply referred to as "rabbit embryo".
（２−２）ウサギ胚のガラス化保存
図１に示す方法に従って、上記（２−１）で得られたウサギ胚をガラス化保存した。具体的には、上記ウサギ胚を、７．５％又は１０％のエチレングリコール（ＥＧ）を含む耐凍剤液中（３０μＬのドロップ）に浸漬して、２３℃で５〜１０分間インキュベートした。インキュベート後の胚を回収して、さらに、ガラス化保存液であるＥＦＳ３０溶液中［３０％（ｖ／ｖ）エチレングリコール、２１％（ｗ／ｖ）フィコール７０、及び０．３５Ｍスクロースを含むＰＢ１液］（３０μＬのドロップ）中に浸漬した。そして、胚を含む５μＬのＥＦＳ３０溶液をクライオチューブ（凍結保存関連製品セラムチューブ、住友ベークライト株式会社製）に移してキャップをした。これらのウサギ胚とＥＦＳ３０溶液とを接触させる工程は、２３℃で合計１分となるように行った。キャップを閉めたクライオチューブを液体窒素に直接浸漬して、ガラス化したウサギ胚凍結し、以下の実験まで液体窒素中で保存した。
(2-2) Vitrification preservation of rabbit embryos The rabbit embryos obtained in (2-1) above were vitrified and preserved according to the method shown in FIG. Specifically, the rabbit embryo was immersed in a freezing agent solution (30 μL drop) containing 7.5% or 10% ethylene glycol (EG) and incubated at 23 ° C. for 5 to 10 minutes. The embryos after incubation are collected, and a PB1 solution containing [30% (v / v) ethylene glycol, 21% (w / v) Ficoll 70, and 0.35 M sucrose in EFS30 solution, which is a vitrification preservation solution. ] (30 μL drop). Then, 5 μL of EFS30 solution containing the embryo was transferred to a cryotube (cryopreservation-related product Serum tube, manufactured by Sumitomo Bakelite Co., Ltd.) and capped. The step of contacting these rabbit embryos with the EFS30 solution was carried out at 23 ° C. for a total of 1 minute. The capped cryotube was immersed directly in liquid nitrogen, vitrified rabbit embryos were frozen and stored in liquid nitrogen until the following experiments.
（２−３）ウサギ胚の融解
図２に示す方法に従って、上記（２−２）で凍結保存したウサギ胚を融解した。具体的には、０．５Ｍのスクロースを含むＰＢ１液（以下、「スクロース液」と称する場合がある）を調製して、２３℃、３７℃、及び５０℃の３つの温度のスクロース液を用意した。そして、上記クライオチューブを液体窒素から取り出してキャップを外し、チューブ内の液体窒素を捨てた後に、２３℃、３７℃、又は５０℃のスクロース液をチューブに加えて、ウサギ胚を融解した。２３℃、３７℃、及び５０℃のスクロース液を用いた場合の融解速度、及び融解に要する時間（カッコ内に記載）はそれぞれ、４，６００℃／分（０．６秒）、６，６００℃／分（０．４秒）、７，８００℃／分（０．３秒）であった。融解したウサギ胚を、２３℃のスクロース液中（３０μＬのドロップ）に５分間浸した後に回収し、１０％ＦＢＳを含むM１９９培地中（２００μＬのドロップ）で培養した。５日間培養後のウサギ胚の卵割率及び胚盤胞期までの発生率を測定した（ｎ＝２）。
(2-3) Thawing of rabbit embryos The rabbit embryos cryopreserved in (2-2) above were thawed according to the method shown in FIG. Specifically, a PB1 solution containing 0.5 M sucrose (hereinafter, may be referred to as “sucrose solution”) is prepared, and sucrose solutions having three temperatures of 23 ° C., 37 ° C., and 50 ° C. are prepared. did. Then, the cryotube was taken out from the liquid nitrogen, the cap was removed, the liquid nitrogen in the tube was discarded, and then a sucrose solution at 23 ° C., 37 ° C., or 50 ° C. was added to the tube to thaw the rabbit embryo. The melting rate and the time required for melting (indicated in parentheses) when using sucrose solutions at 23 ° C, 37 ° C, and 50 ° C are 4,600 ° C / min (0.6 seconds) and 6,600, respectively. It was ° C./minute (0.4 seconds) and 7,800 ° C./min (0.3 seconds). The thawed rabbit embryos were soaked in a sucrose solution at 23 ° C. (30 μL drops) for 5 minutes and then collected and cultured in M199 medium containing 10% FBS (200 μL drops). The cleavage rate of rabbit embryos after culturing for 5 days and the incidence rate up to the blastocyst stage were measured (n = 2).
（２−４）ウサギ胚の生存率及び発生率に及ぼす融解速度の影響
上記（２−３）により得られた結果を図４に示す。図４に示されるように、融解に要する時間が０．６秒であった群（融解温度が２３℃）では、ウサギ胚の卵割率は４０％程度であり、胚盤胞期までの発生率は２０％以下であった。また、融解に要する時間が０．４秒であった群（融解温度が３７℃）では、ウサギ胚の卵割率は７０％程度であり、胚盤胞期までの発生率は５０％程度であった。一方、融解に要する時間が０．３秒であった群（融解温度が５０℃）では、ウサギ胚の卵割率は１００％程度に、胚盤胞期までの発生率は８０％以上までそれぞれ上昇し、無処理群（凍結融解をしていない新鮮ウサギ１細胞期胚）と同程度の高いレベルであった。
以上の結果から、ラットと同様に、ウサギにおいても、融解速度が凍結胚の機能に大きな影響を及ぼすことが明らかとなった。また、５０℃のスクロース液を用いて凍結ウサギ胚を急速融解（７，８００℃／分）することによって、新鮮胚と同等の高い卵割率及び胚盤胞期までの発生率を維持できることが明らかとなった。
(2-4) Effect of melting rate on the survival rate and development rate of rabbit embryos The results obtained by the above (2-3) are shown in FIG. As shown in FIG. 4, in the group in which the time required for thawing was 0.6 seconds (melting temperature was 23 ° C.), the cleavage rate of rabbit embryos was about 40%, and development up to the blastocyst stage. The rate was less than 20%. In the group in which the time required for thawing was 0.4 seconds (melting temperature was 37 ° C.), the cleavage rate of rabbit embryos was about 70%, and the incidence until the blastocyst stage was about 50%. there were. On the other hand, in the group in which the time required for thawing was 0.3 seconds (melting temperature was 50 ° C.), the cleavage rate of rabbit embryos was about 100%, and the incidence rate up to the blastocyst stage was 80% or more. It was elevated and was at a high level comparable to that of the untreated group (fresh rabbit 1-cell stage embryos not thawed).
From the above results, it was clarified that the thawing rate has a great influence on the function of frozen embryos in rabbits as well as in rats. In addition, by rapidly thawing frozen rabbit embryos (7,800 ° C./min) using a sucrose solution at 50 ° C., it is possible to maintain a high cleavage rate and development rate up to the blastocyst stage, which are equivalent to those of fresh embryos. It became clear.
Claims (15)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2019133275A JP7355368B2 (en) | 2019-07-19 | 2019-07-19 | Cryopreservation method for early mammalian embryos |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2019133275A JP7355368B2 (en) | 2019-07-19 | 2019-07-19 | Cryopreservation method for early mammalian embryos |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2021016336A true JP2021016336A (en) | 2021-02-15 |
| JP7355368B2 JP7355368B2 (en) | 2023-10-03 |
Family
ID=74562990
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2019133275A Active JP7355368B2 (en) | 2019-07-19 | 2019-07-19 | Cryopreservation method for early mammalian embryos |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP7355368B2 (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005040073A (en) * | 2003-07-23 | 2005-02-17 | Yoshinori Fukuda | Method and container for freeze-storing reproductive cell |
| WO2006059626A1 (en) * | 2004-12-03 | 2006-06-08 | Nipro Corporation | Device for biosample storage |
-
2019
- 2019-07-19 JP JP2019133275A patent/JP7355368B2/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005040073A (en) * | 2003-07-23 | 2005-02-17 | Yoshinori Fukuda | Method and container for freeze-storing reproductive cell |
| WO2006059626A1 (en) * | 2004-12-03 | 2006-06-08 | Nipro Corporation | Device for biosample storage |
Non-Patent Citations (1)
| Title |
|---|
| SEKI S. ET AL., CRYOBIOLOGY, vol. 81, JPN6023016780, 2018, pages 132 - 137, ISSN: 0005050174 * |
Also Published As
| Publication number | Publication date |
|---|---|
| JP7355368B2 (en) | 2023-10-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Kasai et al. | Survival of mouse morulae vitrified in an ethylene glycol-based solution after exposure to the solution at various temperatures | |
| Kasai et al. | Effects of various cryoprotective agents on the survival of unfrozen and frozen mouse embryos | |
| Mazur | Fundamental aspects of the freezing of cells, with emphasis on mammalian ova and embryos | |
| Nishijima et al. | Effects of type III antifreeze protein on sperm and embryo cryopreservation in rabbit | |
| An et al. | Viable spermatozoa can be recovered from refrigerated mice up to 7 days after death | |
| Michelmann et al. | Cryopreservation of human embryos | |
| Han et al. | Vitrification of rat embryos at various developmental stages | |
| JP3044323B1 (en) | Cell cryopreservation method | |
| Chen et al. | Cryopreservation of tissues and organs: present, bottlenecks, and future | |
| Honaramooz | Cryopreservation of testicular tissue | |
| US8852078B2 (en) | High-throughput and non-invasive method to vitrify porcine embryos | |
| JP7355368B2 (en) | Cryopreservation method for early mammalian embryos | |
| JP2686405B2 (en) | Cryopreservation method of bovine fertilized eggs | |
| Lunardi et al. | Restoring fertility after ovarian tissue cryopreservation: a half century of research | |
| JP2012501187A5 (en) | ||
| Gajda et al. | Oocyte and embryo cryopreservation-state of art and recent developments in domestic animals. | |
| JP2005261413A (en) | Animal embryo freezing and storing liquid and animal embryo freezing and storing method using the same | |
| US20080145930A1 (en) | Methods and compositions for reanimating cryopreserved oocytes | |
| US20080213887A1 (en) | Methods and compositions for cryopreserving oocytes | |
| Otoi et al. | Developmental competence of bovine oocytes frozen at different cooling rates | |
| CN106993605A (en) | A kind of down producing goat freezing seminal fluid dilution formula | |
| US20080113431A1 (en) | Methods and compositions for cryopreserving oocytes | |
| JP3872863B2 (en) | Mammal embryo storage straw | |
| JPH03232441A (en) | Frozen fertilized egg enhanced in conception rate and preparation thereof | |
| Malmir et al. | The Effects of Two Different Freezing Method on Survival and Development of Pronuclear Stage Human Embryos |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20220607 |
|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20230424 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20230508 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20230607 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20230911 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20230913 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 7355368 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |