MC Schieweは、毎年恒例のCDCとSARTデータを分析し、評価する上で彼の統計的な専門知識のためにラスベガスの不妊センターで氏の森ガーナーに感謝したいと思います。また、著者らは、当社の技術力と専門知識の彼の専用サポートと信仰のために、彼らのメディカルディレクター、ロバートE.アンダーソンに感謝したいです。

<ol>
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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+historic+development+and+incorporation+of+four+assisted+reproductive+technologies+shaping+today's+IVF+industry.">The historic development and incorporation of four assisted reproductive technologies shaping today's IVF industry.</a> J. Fertil. In Vitro Reprod. Med. Genet. 4, 2-9 (2016).</li><li>Rall, W. F., Fahy, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ice-free+cryopreservation+of+mouse+embryos+at+-196+degrees+C+by+vitrification.">Ice-free cryopreservation of mouse embryos at -196 degrees C by vitrification.</a> Nature. 313, 573-575 (1985).</li><li>Rall, W. F., Wood, M. J., Kirby, C., Whittingham, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+mouse+embryos+cryopreserved+by+vitrification.">Development of mouse embryos cryopreserved by vitrification.</a> J. Reprod. Fertil. 80, 499-504 (1987).</li><li>Schiewe, M. C., Rall, W. F., Stuart, L. D., Wildt, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+cryoprotectant,+cooling+rate+and+in+situ+straw+dilution+using+conventional+freezing+or+vitrification+for+cryopreserving+sheep+embryos.">Analysis of cryoprotectant, cooling rate and in situ straw dilution using conventional freezing or vitrification for cryopreserving sheep embryos.</a> Theriogenology. 36, 279-293 (1991).</li><li>Lane, M., Schoolcraft, W. B., Gardner, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitrification+of+mouse+and+human+blastocysts+using+a+novel+cryoloop+container-less+technique.">Vitrification of mouse and human blastocysts using a novel cryoloop container-less technique.</a> Fertil. Steril. 72, 1073-1078 (1999).</li><li>Mukaida, T., Oka, C., Goto, T., Takahashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+shrinkage+of+blastocoeles+using+either+a+micro-needle+or+a+laser+pulse+prior+to+the+cooling+steps+of+vitrification+improves+survival+rate+and+pregnancy+outcome+of+vitrified+human+blastocysts.">Artificial shrinkage of blastocoeles using either a micro-needle or a laser pulse prior to the cooling steps of vitrification improves survival rate and pregnancy outcome of vitrified human blastocysts.</a> Hum. Reprod. 21, 3246-3252 (2006).</li><li>Kuwayama, M., Vatja, G., Ieda, S., Kato, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+open+and+closed+methods+for+vitrification+of+human+embryos+and+elimination+of+potential+contamination.">Comparison of open and closed methods for vitrification of human embryos and elimination of potential contamination.</a> Reprod. Biomed. Online. 11, 608-614 (2005).</li><li>Vanderzwalmen, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Births+after+vitrification+at+morula+and+blastocyst+stages:+effect+of+artificial+reduction+of+the+blastocoelic+cavity+before+vitrification.">Births after vitrification at morula and blastocyst stages: effect of artificial reduction of the blastocoelic cavity before vitrification.</a> Hum. Reprod. 17, 744-751 (2002).</li><li>Schiewe, M. C., Marco-Jim&#233;nez, F., AkdemirKo&#231;, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quality+control+factors+influencing+the+successful+and+reliable+implementation+of+oocyte+and+embryo+vitrification.">Quality control factors influencing the successful and reliable implementation of oocyte and embryo vitrification.</a> Cryopreservation. , (2016).</li><li>Stachecki, J. J., Garrisi, J., Sabino, S., Caetano, J. P. J., Wiemer, K., Cohen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+safe,+simple,+and+successful+vitrification+method+for+bovine+and+human+blastocysts.">A new safe, simple, and successful vitrification method for bovine and human blastocysts.</a> Reprod. Biomed. Online. 17, 360-367 (2008).</li><li>Panagiotidis, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open+versus+closed+vitrification+of+blastocysts+from+an+oocyte-donation+programme:+a+prospective+randomized+study.">Open versus closed vitrification of blastocysts from an oocyte-donation programme: a prospective randomized study.</a> Reprod. Biomed. Online. 26, 470-476 (2013).</li><li>Papatheodorou, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open+versus+closed+oocyte+vitrification+system:+a+prospective+randomized+study.">Open versus closed oocyte vitrification system: a prospective randomized study.</a> Reprod. Biomed. Online. 26, 595-602 (2013).</li><li>Schiewe, M. C., Zozula, S., Anderson, R. E., Fahy, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+microSecure+vitrification+(&#181;S-VTF)+for+the+effective+cryopreservation+of+human+embryos+and+oocytes.">Validation of microSecure vitrification (&#181;S-VTF) for the effective cryopreservation of human embryos and oocytes.</a> Cryobiology. 71, 264-272 (2015).</li><li>Schiewe, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroSecure+vitrification+for+oocytes+and+embryos:+optimum+simplicity,+security+and+cost+and+effectiveness+combining+FDA-approved+products.">MicroSecure vitrification for oocytes and embryos: optimum simplicity, security and cost and effectiveness combining FDA-approved products.</a> J. Clin. Embryol. 13, 33-51 (2010).</li><li>Rall, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+affecting+the+survival+of+mouse+embryos+cryopreserved+by+vitrification.">Factors affecting the survival of mouse embryos cryopreserved by vitrification.</a> Cryobiology. 24, 387-402 (1987).</li><li>Seki, S., Mazur, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+warming+rate+on+the+survival+of+vitrified+mouse+oocytes+and+on+the+recrystallization+of+intracellular+ice.">Effect of warming rate on the survival of vitrified mouse oocytes and on the recrystallization of intracellular ice.</a> Biol. Reprod. 79, 727-737 (2008).</li><li>Schiewe, M. C., Stringfellow, D. A., Givens, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+hygiene+and+quality+control+practices+in+an+embryo-production+laboratory.">General hygiene and quality control practices in an embryo-production laboratory.</a> Manual of the International Embryo Transfer Society: A Procedural Guide and General Information for the Use of Embryo Transfer Technology, Emphasizing Sanitary Precautions. , (2008).</li><li>Whitney, J. B., Nugent, N. L., Anderson, R. E., Schiewe, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+center+validation+of+routine+blastocyst+biopsy+implementation.">Single center validation of routine blastocyst biopsy implementation.</a> J. Asst. Reprod. Genet. , (2016).</li><li>Liebermann, L., Conaghan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+collapse+prior+blastocyst+vitrification:+improvement+of+clinical+outcomes.">Artificial collapse prior blastocyst vitrification: improvement of clinical outcomes.</a> J. Clin. Embryol. 16, 107-118 (2014).</li><li>Parmegiani, L., Tatone, C., Cognigni, G. E., Bernardi, S., Troilo, E., Arnone, A., Maccarini, A. M., Di Emidio, G., Vitti, M., Filicori, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+warming+increases+survival+of+slow-frozen+sibling+oocytes:+a+step+towards+a+single+warming+procedure+irrespective+of+the+freezing+protocol.">Rapid warming increases survival of slow-frozen sibling oocytes: a step towards a single warming procedure irrespective of the freezing protocol.</a> Reprod Biomed Online. , (2014).</li></ol>

MC Schieweは、革新的なクライオ企業への科学諮問委員会のメンバーとして、カリフォルニア精子バンクにテクニカルディレクターとして働きます。このビデオ記事の生産は大喝采豊饒によって支払われています。著者には、競合する金融契約または配偶子および胚のガラス化に関する開示する利害の衝突を持っていません。

今日では、完全な胚盤胞の生存率（&gt; 95％）を達成し、新鮮胚のと同様の移植の成功を達成するための高い期待があります。いくつかのグループには、ガラス固化体胚移植サイクルの出生率は、無傷の凍結保存した胚は、健康な、非ホルモンで刺激された子宮に移植された新鮮胚盤胞よりもさらに高い、おそらくあることを示唆しています。我々のデータは明らかにガラス化が効果的かつ確実に新鮮胚の生存率を維持していることを示しています。さらに、我々は、無菌ことを証明した、MicroSecureガラス化（μS-VTF）と呼ばれる閉じた方法は、に匹敵するか、IVF産業で使用される市販の標準（ すなわち、オープンデバイス・システム）以上の最適な結果を達成することができます。 バリエーションは、ガラス化デバイス/多数の方法を使用して、個人間の技術的な再現性と信頼性に関連しています。今度は、これはinconsiをもたらしましたガラス化を適用するプログラム間stencies。したがって、デバイスの親しみは、実験室での習熟度と成功の成果に関する重要な要因であることは驚くべきことではありません。これは、プログラム間の再現性が問題となり得る理由を説明し、「技術的な署名」11のこの概念です。ダース以上の商業機器の開発は、この現象を複雑にするだけでなく、品質管理上の問題を作成していました。幸いなことに、μS-VTF、ラピッドIのように、ガラス化システムを閉じ、そしてVitriSafeは、今のデバイス・システム12、13、14を開くことが同等に有効であることが証明されています。 MicroSecure VTFは心の中で技術的な使いやすさ、信頼性、およびクライオセキュリティで開発された小説、無菌ガラス化技術です。 2以前に承認され、FDAに準拠したデバイスの使用を組み合わせることにより、非商用のガラス固化システムワットであります特殊なデバイスとは対照的に、確立された低コストを有することの明確な利点第i。また、独自の改竄防止や内在化2色ラベリングシステムと同様に、多数の他の品質管理の利点は、作ったμS-VTF魅力的なグローバルオプション11。非商用VTF装置として、しかし、その広範な業界のアプリケーションがゆっくりと進化しています。のみ、その安全性、セキュリティ、および臨床有効性の継続的な出版物を通じて利用をμS-VTFゲイン成長します。 インナー、非ウィッキング疎水性プラグで製造オリジナルの0.3-mLの胚のストローを取得することができないことに直面したときに2014年8月では、我々は確実にμS-VTF方法を変更することができました。要するに、彼らの綿-PVPプラグを持つ新しい0.3 mLの精液/胚のストローを効果的に適合させました。これは、単にこのように接触し、流体ウィッキングを防止し、綿栓の前に内部シールを作成することによって達成されましたオープンエンドVTFの先端（ すなわち、胚や卵を含むflexipette）。 40 mmのIDロッドはアクセスできない場合はさらに、軽く30 mmの棒に関連付けられている浮力の問題は、2つのボールベアリングを使用してcounterweightedすることができます。 これらの追加の手順は、技術がわずかに少ないシンプル作ったが、まだ非常に効果的です。その正倍数性状態は、遺伝学のレポートで確認されるまで、生検の胚盤胞は、典型的には、個別にガラス化しているように今日では、経済性と有効性は、ますます重要な関心事になっています。確認された非生存異数性のステータスを持つ胚盤胞は、通常、数週間以内に、患者の同意を得て、破棄されます。したがって、VTFデバイスの大部分（&gt; 50％）は、商用デバイスを使用する場合にエスカレート年間コストを引き起こし、短期保管後に廃棄されます。全体的に、μS-VTFは、人間の胚盤胞の凍結保存のために、非常に効果の高い信頼性、および反復可能な手順です。優れた品質・制御設計securelyのラベルや安全に回復の障害を排除し、胚のガラス化への普遍的なアプローチとして、システムの使用を正当化後の温暖化の生存および生存度を、最適化しながら、胚/卵母細胞を格納します。

ガラス化は、細胞質内精子注入の開発以来、 体外受精（IVF）業界で単一の最も影響力の生殖補助技術です。今日では、胚盤胞は、以前に、従来の低速凍結方法1に関連付けられた胚の生存率の損失なしに凍結保存します。信頼性の高いポスト温暖化胚の生存と、不妊の業界は、従来の新鮮胚移植よりも同等以上の妊娠の転帰をもたらす凍結保存した胚移植サイクルの好ましい使用に転換されます。胚盤胞の生検および着床前遺伝子スクリーニング（PGS）との関連では、ガラス化が正倍数体の単一胚移植2、3を介して健康的な出生の成果を最適化するために不可欠な臨床ツールとなっています。 マウス胚のガラス化は、1980年代半ば4で開発されました</SUP&gt; 5年と1990年6により畜産に適応。ガラス化溶液は、氷結晶形成を損傷のない、準安定glasseous状態を形成することを前提に基づいて、より効率的に胚の完全な細胞の完全性を維持することが証明されています。興味深いことに、ヒトの発生学へのガラス化の有望な受け入れは、21世紀までに実現することを開始しませんでした。ガラス化の利用を促進する初期の刊行物は、ユニークな「オープン」システム・デバイス7、8、9の開発と一致しました。それは遅い胚盤胞の凍結の改善にも発生した時に来たとしてしかし、臨床診療へのガラス化の採用は、遅かったです。成功し、従来の遅い速度の凍結は、ガラス化に加えて、だけでなく、incorporatで、胚培養システムの改善と整列させましたその後、栄養外胚葉の両方の全体的な生存を強化し、胞胚-崩壊のアプローチのイオン、注入10。 最後の10年間で、ガラス固化技術が急速に従来の凍結慣行に取って代わりました。大幅に、これは特殊なガラス化デバイスの開発によるものでした。これらのデバイスのいくつかは、IVF産業11内で使用されているデバイスに固有の設計上の欠陥を導入することにより、臨床ガラス化の全体的な安全性、効率性及び有効性をハンディいます。実際に、異なるデバイスのニュアンスは、一般に「技術的な署名」12と呼ばれるプログラム間の重要な技術的変化を、ご紹介します。このように、科学雑誌は、可視化実験のジャーナル（Joveの）のように、結果のばらつきを低減するのに役立ちます技術的な詳細を、実証するための貴重な資源としての役割を果たすことができます。もう一つの継続的な問題は、sであります青梅胎生学者は、「オープンガラス化システム」における胚または卵母細胞の「超急速冷却（液体窒素（LN 2）を持つ、すなわち、直接胚の接触は）最適化するための前提条件であるという主張に基づいて、今日でも、誤解され続け成功率。」明らかに、この信念は、無菌の実証済みの成功に基づいて、不正確であるシステム13、14、15を閉じました。 ガラス化のcryobiological原理に基づいて、ガラス化の効果は速度16、17、18の冷却に比べて温暖化レートに、より大きく依存します。一般に、使用されるガラス化装置の独立した、加温速度は、高い生存率を保証するために冷却速度を超えなければなりません。高い温暖化率は、任意の氷の成長（ すなわち、recrの機会を最小限に抑えます温暖化の失透フェーズ中にクライオソリューション）中の有核の不純物のystallization。確かに、ガラス化溶液（ すなわち、タイプおよび使用される凍結保護剤の濃度）の安定性は、交絡効果を有することができるが、これは別のパブリケーション11で対処されています。冷却加温速度の問題を考慮すると、MicroSecureガラス化（μS-VTF）ガラス化の品質管理の側面を最適化し、安価な、非商用、FDA準拠法として2008年に開発されました。それは、耐タンパ性を、内在化、二重色の標識を提供していることで独特でした。さらに、ロードおよびピペッティングのために使用される滅菌flexipetteに直接胚を保存する（ すなわち、二次デバイス表面にピペッティングせず）によると、完全に溶接シールが自動化されたシーラーを使用して、アイオノマー樹脂製のストローを使用することにより、技術的な変動が効果的に除去されています。 Cを評価する場合潜在的な使用のためのガラス固化装置のompleteness、などの考慮すべきであるいくつかの品質管理の要因がある：1） 標識化の可能性 -CANラベルが確実に接着させることは？彼らは不正開封防止していますか？彼らは、デュアル色識別の可能性を提供していますか？それは、二次ラベルを必要としない、とラベルは簡単に記録保持の目的のために取り外すことができる（ すなわち、患者確認）後の温暖化？ 2） 技術的な使いやすさ -CAN胚は簡単にタイムリーにデバイスに/にロードされ、簡単に識別され、後の温暖化を追跡することが？ 3） 手続きシンプルさ/再現性は、技術者（内部）とプログラム（外部）間の変動を最小限に抑えることが容易に再現することが可能ガラス化法オファーシンプルさと信頼性を、-Does？ 4）LN 2の記憶容量を容易にかつ安全に取り扱われ、識別されるデバイスをれる-CAN？そのSTOです効率的な潜在的な空間を激怒？無菌閉鎖システムなどの物理的な損傷または可能な汚染物質からのデバイスのオファーのセキュリティと安全性をしていますか？ 5） 回復の可能性/耐障害 -Isデバイス胚の保証回復における潜在的な問題になりやすいデザイン、そして、彼らは確実にガラス化し、完全な細胞の完全性ポスト温暖化を維持するのだろうか？後者は特定の品質上の問題、回収率は、実際には、驚くべきことに公表された報告に最小化されています。これは、一般に、典型的に、良好な生存率の不利な結果（ すなわち、失われた胚または卵子を）隠すことによって行われます。一貫性のない回復（&lt;99％）になりやすい任意のデバイスは真剣に欠陥があると手続き負債を構成しています。 私たちの無菌は、μS-VTF方法は戦略的に各品質管理対策を考慮して開発された閉じました。しかし、優れた臨床的成功と検証14の5年後、手続きはトンを持っていましたO修正すること。 （疎水性プラグを有する）元の0.3-mLの胚のストローは、IVF業界から除去し、標準的な綿/ PVP栓を有する0.3-mLの精液ストロー（ すなわち、精液/胚のストローとして再度ラベル付け）に置き換えられました。この手続き論文では、安全に、簡単に、かつ効果的μS-VTFを実装するために必要な具体的な手順と戦略の概要を説明します。代替理想的なわらのコンテナが臨床検査室に戻って再導入されるようにさらに、我々はそのような時間まで、確実に供給制限を考慮するために必要な修正（複数可）をハイライト表示します。

<table><tbody><tr><td>Aluminum Cane</td><td>IVM</td><td>XC055</td><td></td></tr><tr><td>Ball bearings, 3/32&quot;</td><td>VXB.com</td><td>KIT15977</td><td>stainless steel</td></tr><tr><td>CBS semen/embryo straw, 0.3 mL</td><td>CryoBioSystems</td><td>25292</td><td>individual sterile</td></tr><tr><td>Color, ID rods, 30 mm</td><td>CryoBioSystems</td><td>019021-26</td><td>weighted</td></tr><tr><td>Culture tubes, 15 mL</td><td>Falcon</td><td>352099</td><td>Conical</td></tr><tr><td>Culture tubes, 10 mL</td><td>Falcon</td><td>352057</td><td>Snap-cap</td></tr><tr><td>Cryosleeves</td><td>Nalgene</td><td>5016-001</td><td></td></tr><tr><td>Filter, 250 mL</td><td>Fisher Sci.</td><td>09-740-2A</td><td>0.22 &amp;mu;m</td></tr><tr><td>Flasks, Tissue Culture 50 mL</td><td>Falcon</td><td>353014</td><td></td></tr><tr><td>Flexipettes, 300 &amp;mu;m ID</td><td>Cook Med.</td><td>K-FPIP-1300-10BS-5</td><td>Sterile, 20/pack</td></tr><tr><td>Forcep, Large</td><td>Miltex</td><td>6-30TC</td><td></td></tr><tr><td>Forcep, Splinter - fine</td><td>Miltex</td><td>17-305</td><td></td></tr><tr><td>Goblet</td><td>IVM</td><td>PA003</td><td></td></tr><tr><td>Heat Sealer, SYMS 1</td><td>CryoBioSystems</td><td>16399</td><td>110 V or 220 V with adapter</td></tr><tr><td>Hepes-buffered media&amp;nbsp;</td><td>Life Global or</td><td>LGGH-100; 100&amp;nbsp;mL, or</td><td>stored at 2 - 8 &amp;ordm;C</td></tr><tr><td></td><td>Irvine Scientific</td><td>H-HTF; 90126; 100 mL</td><td>with non-essential AA&#039;s</td></tr><tr><td>Labels, Cryo</td><td>GA International</td><td>CL-23T1</td><td>Various colors</td></tr><tr><td>Liquid Nitrogen Tank, 40&amp;nbsp;L</td><td>MVE or Taylor Warton</td><td>various</td><td>liquid storage&amp;nbsp;</td></tr><tr><td>LN&lt;sub&gt;2&lt;/sub&gt;; Dewar flask, 0.5&amp;nbsp;L</td><td>Hampton Research</td><td>HR4-695</td><td>Stainless steel</td></tr><tr><td>6-well Custer Dishes</td><td>Biogenics</td><td>015/020</td><td>plasticware by case</td></tr><tr><td>Pipette Bulb, Micro Cap</td><td>Drummond</td><td>Fisher#13681451</td><td>Hole on bulb apex</td></tr><tr><td>Petri Dishes, 35 mm</td><td>Falcon</td><td>351006</td><td></td></tr><tr><td>Petri Dishes, 58 mm</td><td>Nunc</td><td>150288</td><td></td></tr><tr><td>Petri Dishes, 100 mm</td><td>Falcon</td><td>351029</td><td></td></tr><tr><td>Pipette Tips, ART long</td><td>Fisher Sci.</td><td>02-707-80</td><td>10 - 100 &amp;mu;L</td></tr><tr><td>Pipet Aid</td><td>Drummond or Falcon</td><td>various</td><td>rechargeable</td></tr><tr><td>Pipetting Device, Stripper</td><td>Cooper Surgical</td><td>MXL3-STR</td><td></td></tr><tr><td>Pipettes, Serological 1&amp;nbsp;mL</td><td>Falcon</td><td>357521</td><td></td></tr><tr><td>Pipettes, Serological 2&amp;nbsp;mL</td><td>Falcon</td><td>357507</td><td></td></tr><tr><td>Pipettes, Serological 5&amp;nbsp;mL</td><td>Falcon</td><td>357543</td><td></td></tr><tr><td>Pipettes, Serological 10&amp;nbsp;mL</td><td>Falcon</td><td>357551</td><td></td></tr><tr><td>Scissors, Surgical Mayo</td><td>Miltex</td><td>5-SC-16</td><td></td></tr><tr><td>Stereomicroscope</td><td>Nikon, Olympus, Leica</td><td>various</td><td></td></tr><tr><td>Sterile Gauze pads, 4&quot; x 4&quot;</td><td>Kendall Healthcare</td><td>6939</td><td></td></tr><tr><td>Synthetic serum</td><td>Life Global, or</td><td>LGPS-20; 20 mL, or</td><td>stored at 2 - 8 &amp;ordm;C</td></tr><tr><td></td><td>Irvine Scientific</td><td>SS-99193; 12 x 10 mL</td><td>purchase low endotoxin lot</td></tr><tr><td>Sucrose</td><td>Sigma Chemical Co.</td><td>#S9378</td><td>Aliquot into 50&amp;nbsp;mL flasks, 1 year;&lt;br/&gt;
17.1 g/flask + Medium to 50 mL;&lt;br/&gt;
makes a 1 M solution;&lt;br/&gt;
Filter with 0.22 &amp;micro;m unit</td></tr><tr><td>Timer</td><td>Nalgene</td><td>5016-001</td><td></td></tr><tr><td>Thawing Solution</td><td>Innovative Cryo Enterprises</td><td>BL-TS (&amp;le;1.0 M Sucrose)</td><td>T1, T2, T3, T4; tored at 2 - 8 &amp;ordm;C for &amp;le;1 month after opening</td></tr><tr><td>Vitrification Solution*,**</td><td>Innovative Cryo Enterprises</td><td>BL-VS (&amp;ge;7.9 M [Glycerol/EG])</td><td>V1, V2, V3; stored at 2 - 8 &amp;ordm;C for &amp;le;1 month after opening</td></tr><tr><td colspan="4">* Non-permeating cryoprotective additives may include: sucrose, ficoll and sodium hyaluronate&amp;nbsp;</td></tr><tr><td colspan="4">** Other commercial preparations are typically ethylene glycol (EG)/dimethyl sulfoxide (DMSO; 30% v/v; 4.8 M), but could be EG/propylene glycol (32% v/v; 5.2 M). Mixed solutions are typically used to reduce cryo-toxicity concerns of a high molar solution. Commercial solutions typically include an ES and VS solution. The formulation of commercial preparation is typically proprietary property.</td></tr></tbody></table>

modified microsecure vitrification: a safe, simple and highly effective cryopreservation procedure for human blastocysts

臨床胚ガラス化は、「オープン」システム（ すなわち、直接LN 2接点）での超急速冷却がガラス化の成功を最適化するために必要であったことを21世紀のユニークなガラス化装置の開発をし、誤解と進化しました。冷却速度の重要性を取り巻くドグマは、重要な品質制御因子（ 例えば、使いやすさ、再現性、信頼性、ラベリングセキュリティ、およびストレージの安全性を）無視技術的変化へとガラス化装置の作成の対象と危険な慣行につながりました。イントラおよびインター技師変動を最小限に抑えることを目的と、安全・安心、再現性、および信頼性の高いμS-VTF法の開発のために許可された他の機器の品質管理の欠陥を理解します。内在化、2色、改ざん-pの1）0.3-mLのアイオノマー樹脂胚わら：同様に重要な、それは2つの既存のFDA準拠デバイスの可用性を組み合わせました反復可能な溶接シール可能性のある屋根の標識;直接非常に効果の高いグローバルなガラス化デバイスを作成するために、胚（複数可）をロードすると、2）短く、一般的に使用され、300μmのID滅菌flexipettes。他の無菌と同様に、ガラス固化システム（ 例えば、高セキュリティガラス化（HSV）、ラピッドI、およびVitriSafe）効果的に生殖医療で使用される閉じ、microSecureガラス化（μS-VTF）が、それが高いポスト温暖化の生存と妊娠を達成することができることを証明しましたそのシンプルさへのこだわり、そして縮小技術的な変化と成果。内部の疎水性のプラグを含む0.3 mLの胚のストローは、商業的に綿ポリビニルピロリドン（PVP）のプラグを有する標準精液ストローで置換したが、溶接密封を確実にするために、そのアイオノマー樹脂組成物を維持しました。しかし、綿プラグが接触した際にflexipettesの流体胚の内容を吸い上げることができます。修正されたμS-VTF方法は、追加の内部溶接部を含むように適合されましたデバイスの負荷側のプラグの前に密封します。高い生存率（&gt; 95％）と妊娠率が今日続けるようμS-VTF手順に追加された技術的なステップは、その成功のアプリケーションに影響を与えていません。

1316が（98.1パーセント）が生存した1341ガラス固化体胚盤胞は、（100％）2014年6月に2012年の間に温め、1341胚を回収しました。生検胚盤胞は、99.5％の生存上で経験しました。主に単一の転送時には、遺伝的にテストされ、正倍数体、ビトリファイド胚、我々は米国で最も高い注入率と出生率のいくつかを達成することができました、年齢の独立した（ 図3）20、センターによって報告された最近の国の統計によると疾病管理生殖補助医療学会（ 表1、表 2）のために。単独で凍結保存されたサイクルのための良好な予後患者集団（未満35歳）（ 表1）を評価する 、私たちの研究室では、両方の注入率の全国平均（ すなわち、妊娠を確立するために胚の効率）を上回りましたし、最終的に出生率を生きます<html> 30％を超えることもできます。さらに、70の研究・承諾し、再ビトリファイド異数体胚盤胞を使用して、半ば2014年に私たちの修正されたストレージ・ストローの初期確認/検証は、回収率100％と100％の生存率をもたらしました。 <img alt="図1" src="/files/ftp_upload/54871/54871fig1.jpg" /> 1. MicroSecure VTFセットアップ図 。 VTF皿（A）は異なる、番号保持液滴に配置する前に、3洗浄液滴を利用するガラス化溶液の3つの異なる列で組み立てられます。また、短縮VTFのヒント（ すなわち、300μmのIDのflexipettes）を有する個々のピペットを確保し、秩序ある使用のために回転させることができる発泡スチロールのチューブラック（B）、で構成されています。代表的な使い捨てのマイクロピペットの電球のピペットアセンブリは発泡スチロールのラックに載っていることに注意してください。<html> ARGET = &quot;_空白&quot;&gt;この図の拡大版をご覧になるにはこちらをクリックしてください。 <img alt="図2" src="/files/ftp_upload/54871/54871fig2.jpg" /> 図2は、 胚盤胞評価システムを変更しました。私たちの修正されたガードナー胚盤胞グレーディングシステムは、胚盤胞の生検を容易にするために、TE細胞の誘導さヘルニアを占めています。孵化胚盤胞（C）が 50％以上ヘルニアを有している：（グレード4 =最大ヘルニア50％B）：（グレード3 = 10％未満のヘルニアA）および拡張胚盤胞修飾は完全胚盤胞の早期ハッチングを考慮します16。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54871/54871fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> 54871fig3.jpg &quot;/&gt; 3.比較妊娠成果図。累積1.5年間隔で、妊娠のデータは、ガラス固化体温め正倍数体胚盤胞を転送の効果を評価するために比較された非PGSサイクルと比較した（n = 172サイクル/ 144転送）は、主に新鮮な関与た（n = 160サイクル/ 153転送） 18を転送します。我々の実験のために、このデータは、μS-VTF手順によってガラス化胚盤胞の生検は、それらの生存能力を維持することが明らかになりました。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54871/54871fig3large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>情報元</td><td> ＃サイクル</td><td>移植胚の＃を意味します</td><td>注入率（％） </td><td>ライブ出生率（％） </td></tr><tr><td> NBラボ* </td><td> 144 </td><td> 1.2 </td><td> 74.00パーセント</td><td> 74.50パーセント</td></tr><tr><td>全国平均</td><td> 20423 </td><td> 1.7 </td><td> 39.60パーセント</td><td> 44.10パーセント</td></tr><tr><td colspan="5"> ※データは2008年にmicroSecureガラス化手順を設立し、現在オベーション不妊研究所（NBラボ）を使用して、オレンジ郡豊饒、南カリフォルニア不妊センターと生殖医療クリニックのための南カリフォルニアセンターの2014年のパフォーマンスに基づいて平均しました。 </td></tr></tbody></table> 表1 2014年CDCは生殖統計を支援します。 3から35歳未満の女性の凍結胚移植妊娠データが450以上報告クリニックの米国2013年全国平均と比較して、私たちのNBラボを使用して、医師の診療所を報告します。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td colspan="2"> SART全国平均</td><td></td><td></td><td></td></tr><tr><td>クリニック - 国家</td><td>女性たち</td><td>女性たち</td><td>女性たち</td><td>女性たち</td></tr><tr><td></td><td> &lt;35歳</td><td> 35-37歳</td><td> 38-40歳</td><td> 41-42歳</td></tr><tr><td> SCCRM-CA * </td><td> 63.1パーセント</td><td> 58.3パーセント</td><td> 40.6％ </td><td> 32.3パーセント</td></tr><tr><td> CCRM-CO * </td><td> 64.7パーセント</td><td> 61.5パーセント</td><td> 40.4パーセント</td><td> 32.2パーセント</td></tr><tr><td> RMA-NJ * </td><td> 63.2パーセント</td><td> 59.7パーセント</td><td> 34.6％ </td><td> 18.7％ </td></tr><tr><td>全国平均</td><td> 48.6パーセント</td><td> 38.3パーセント</td><td> 24.3％ </td><td> 12.3％ </td></tr><tr><td colspan="5">生殖医療のためのSCCRM-南カリフォルニアセンター。生殖医療のためのためのCCRMコロラドセンター;そして、RMA-Reproductive医学アソシエイツ</td></tr><tr><td colspan="5"> *これらの大手クリニックはすべて、主に移植胚あたりの出生の成功を最適化するために、患者ケアの彼らの標準的な実践に、着床前遺伝子検査に関連して、ガラス化した胚移植サイクルを実装しています。 </td></tr></tbody></table> 他のいくつかの尊敬のプログラムと、だけでなく、SARTと対比ニューポートビーチ、カリフォルニア州で私たちのオベーション豊饒ラボを使用してSCCRMクリニックのための生殖補助医療のための協会によって報告されるように表2 2014累積出生率、（SART）、全国平均。

Watch this Scientific Journal Video about Modified MicroSecure Vitrification: A Safe, Simple and Highly Effective Cryopreservation Procedure for Human Blastocysts at JoVE.com

Modified MicroSecure Vitrification: A Safe, Simple and Highly Effective Cryopreservation Procedure for Human Blastocysts

MicroSecure Vitrification was developed as a non-commercial, aseptic closed vitrification device system that is compliant with FDA good manufacturing and tissue-handling practices. Due to the withdrawal of hydrophobic plugged embryo straws from the industry, the vitrification procedure was modified to include an inner seal before the standard internal cotton plug.

修正されたMicroSecureガラス化：人間の胚盤胞のための、安全なシンプルで非常に効果的な凍結保存手順

Clinical embryo vitrification evolved with the development of unique vitrification devices in the 21st century and with the misconception that ultra-rapid ...

modified-microsecure-vitrification-safe-simple-highly-effective

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

11.8K ビュー.  Ovation Fertility. MicroSecure Vitrification was developed as a non-commercial, aseptic closed vitrification device system that is compliant with FDA good manufacturing and tissue-handling practices. Due to the withdrawal of hydrophobic plugged embryo straws from the industry, the vitrification procedure was modified to include an inner seal before the standard internal cotton plug.

ビデオ: Modified MicroSecure Vitrification: A Safe, Simple and Highly Effective Cryopreservation Procedure for Human Blastocysts

Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal and postnatal consequences. To ensure the innocuousness of ART applications, studies in animal models are necessary. In addition, as a last step, embryo development studies require evaluation of their capacity to develop full-term healthy offspring. Here, embryo transfer to the uterus is indispensable to perform any ARTs-related experiment.
The rabbit has been used as a model organism to study mammalian reproduction for over a century. In addition to its phylogenetic proximity to the human species and its small size and low maintenance cost, it has important reproductive characteristics such as induced ovulation, a chronology of early embryonic development similar to humans and a short gestation that allow us to study the consequences of ART application easily. Moreover, ARTs (such as intracytoplasmic sperm injection, embryo culture, or cryopreservation) are applied with suitable efficiency in this species.
Using the laparoscopic embryo transfer technique and the cryopreservation protocol presented in this article, we describe 1) how to transfer embryos through an easy, minimally invasive technique and 2) an effective protocol for long-term storage of rabbit embryos to provide time-flexible logistical capacities and the ability to transport the sample. The outcomes obtained after transferring rabbit embryos at different developmental stages indicate that morula is the ideal stage for rabbit embryo recovery and transfer. Thus, an oviductal embryo transfer is required, justifying the surgical procedure. Furthermore, rabbit morulae are successfully vitrified and laparoscopically transferred, proving the effectiveness of the described techniques.

Assisted reproductive techniques (ARTs) are in continuous evaluation to improve outcomes and reduce the associated risks. This manuscript describes a minimally invasive embryo transfer procedure with an efficient cryopreservation protocol that allows the use of rabbits as an ideal animal model of human reproduction.

ウサギモデルにおける最適胚期における低侵襲胚移植と胚ガラス化

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal ...

発生生物学

Human Blastocyst Biopsy and Vitrification

Blastocyst biopsy is performed to obtain a reliable genetic diagnosis during IVF cycles with preimplantation genetic testing. Then, the ideal workflow entails a safe and efficient vitrification protocol, due to the turnaround time of the diagnostic techniques and to transfer the selected embryo(s) on a physiological endometrium in a following natural cycle. A biopsy approach encompassing the sequential opening of the zona pellucida and retrieval of 5-10 trophectoderm cells (ideally 7-8) limits both the number of manipulations required and the exposure of the embryo to sub-optimal environmental conditions. After proper training, the technique was reproducible across different operators in terms of timing of biopsy (~8 min, ranging 3-22 min based on the number of embryos to biopsy per dish), conclusive diagnoses obtained (~97.5%) and live birth rates after vitrified-warmed euploid blastocyst transfer (&#62;40%). The survival rate after biopsy, vitrification and warming was as high as 99.8%. The re-expansion rate at 1.5 h from warming was as high as 97%, largely dependent on the timing between biopsy and vitrification (ideally &#8804;30 min), blastocyst morphological quality and day of biopsy. In general, it is better to vitrify a collapsed blastocyst; therefore, in non-PGT cycles, laser-assisted artificial shrinkage might be performed to induce embryo collapse prior to cryopreservation. The most promising future perspective is the non-invasive analysis of the IVF culture media after blastocyst culture as a putative source of embryonic DNA. However, this potential avant-garde is still under investigation and a reliable protocol yet needs to be defined and validated.

Blastocyst biopsy and vitrification are required to efficiently conduct preimplantation genetic testing. An approach entailing the sequential opening of the zona pellucida and retrieval of 7-8 trophectoderm cells in day 5-7 post-insemination limits both the number of manipulations required and the exposure of the embryo to sub-optimal environmental conditions.

ヒト胚盤胞生検とガラス化

Blastocyst biopsy is performed to obtain a reliable genetic diagnosis during IVF cycles with preimplantation genetic testing. Then, the ideal workflow ...

Minimum Volume Vitrification of Immature Feline Oocytes

In wild animals&#8217; conservation programs, gamete banking is crucial to safeguard genetic resources of valuable individuals and rare species and to promote biodiversity preservation. In felids, most species are threatened with extinction, and domestic breeds are used as a model to increase the efficiency of protocols for germplasm banking. Among oocyte cryopreservation techniques, vitrification is more and more popular in human and veterinary assisted reproduction. Cryotop vitrification, which was at first developed for human oocytes and embryos, has demonstrated to be well-suited for cat oocytes. This method offers several advantages, such as the feasibility in field conditions and the speed of the procedure, which can be helpful when several samples need to be processed. However, the efficiency is strongly dependent on the operator&#8217;s skills, and intra- and inter-laboratory standardization are needed, as well as personnel training. This protocol describes minimum volume vitrification of immature feline oocytes on a commercial support in a step by step field-friendly protocol, from oocyte collection to warming. Following the protocol, preservation of oocyte integrity and viability at warming (as high as 90%) can be expected, although there is still room for improvement in post-warming maturation and embryonic development outcomes.

This manuscript describes a protocol for the minimum volume vitrification of immature cat oocytes with laboratory-made media on commercial supports. It covers every step from oocyte isolation from ex vivo gonads to vitrification and warming.

未熟ネコの最小体積ガラス化

In wild animals&#8217; conservation programs, gamete banking is crucial to safeguard genetic resources of valuable individuals and rare species and to ...

Vitrification of In Vitro Matured Oocytes Collected from Adult and Prepubertal Ovaries in Sheep

In livestock, in vitro embryo production systems can be developed and sustained thanks to the large number of ovaries and oocytes that can be easily obtained from a slaughterhouse. Adult ovaries always bear several antral follicles, while in pre-pubertal donors the maximal numbers of oocytes are available at 4 weeks of age, when ovaries bear peak numbers of antral follicles. Thus, 4 weeks old lambs are considered good donors, even if the developmental competence of prepubertal oocytes is lower compared to their adult counterpart. 
Basic research and commercial applications would be boosted by the possibility of successfully cryopreserving vitrified oocytes obtained from both adult and prepubertal donors. The vitrification of oocyte collected from prepubertal donors would also allow shortening the generation interval and thus increasing the genetic gain in breeding programs. However, the loss of developmental potential after cryopreservation makes mammalian oocytes probably one of the most difficult cell types to cryopreserve. Among the available cryopreservation techniques, vitrification is widely applied to animal and human oocytes. Despite recent advancements in the technique, exposures to high concentrations of cryoprotective agents as well as chilling injury and osmotic stress still induce several structural and molecular alterations and reduce the developmental potential of mammalian oocytes. Here, we describe a protocol for the vitrification of sheep oocytes collected from juvenile and adult donors and matured in vitro prior to cryopreservation. The protocol includes all the procedures from oocyte in vitro maturation to vitrification, warming and post-warming incubation period. Oocytes vitrified at the MII stage can indeed be fertilized following warming, but they need extra time prior to fertilization to restore damage due to cryopreservation procedures and to increase their developmental potential. Thus, post-warming culture conditions and timing are crucial steps for the restoration of oocyte developmental potential, especially when oocyte are collected from juvenile donors.

The protocol aims at providing a standard method for the vitrification of adult and juvenile sheep oocytes. It includes all the steps from the preparation of the in vitro maturation media to the post-warming culture. Oocytes are vitrified at the MII stage using Cryotop to ensure the minimum essential volume.

羊の成人および前頭前卵巣から採取された体外期の成熟卵母細胞のガラス化

In livestock, in vitro embryo production systems can be developed and sustained thanks to the large number of ovaries and oocytes that can be easily ...

Morphometric Protocol for the Objective Assessment of Blastocyst Behavior During Vitrification and Warming Steps

This article describes the noninvasive method of blastocyst morphometry based on time-lapse microphotography for the accurate monitoring of a blastocyst&#39;s volume changing during individual phases before and after vitrification. The method can be useful in searching for the most optimal timing of blastocyst exposure to different concentrations of cryoprotectants by observing blastocyst shrinkage and re-expansion in different pre- and post-vitrification phases. With this methodology, the blastocyst vitrification protocol can be optimized. For a better demonstration of the usefulness of this morphometric method, two different blastocyst preparation protocols for vitrification are compared; one with using an artificial blastocoel collapsing and one without this intervention before vitrification. Both blastocysts&#39; volume changes are followed by time-lapse microphotography and measured by photo-editing software tools. The measurements are taken every 20 seconds in previtrification phases and every 5 minutes in the post-warming period. The changes of the blastocyst dimensions per time unit are presented graphically in line diagrams. The results show a long equilibration previtrification phase in which the intact blastocyst first shrinks and then slowly refills the blastocoel, entering vitrification with a fluid-filled blastocoel. The artificially collapsed blastocyst remains in its shrunken stage through the entire equilibration phase. During the vitrification phase, it also does not change its volume. Since the blastocyst morphometry shows a constant volume of the artificially collapsed blastocysts during the previtrification step, it seems that this stage could be shorter. The described protocol provides many additional comparative parameters of blastocyst behavior during and after cryopreservation on the basis of the speed and intensity of the volume changes, the number of partial blastocoel contractions or total blastocyst collapses, and the time to a total blastocoel re-expansion or the time to hatching.

Here we present a time-lapse morphometric protocol to follow the intensity of blastocyst shrinkage and re-expansion during previtrification interventions and post-warming recovery. The application of the protocol is possible in in vitro fertilization laboratories equipped with time-lapse microscopes and is recommended in the development of an optimal blastocyst vitrification method.

ガラス化と地球温暖化の手順中に胚盤胞の行動の客観的評価のための形態のプロトコル

This article describes the noninvasive method of blastocyst morphometry based on time-lapse microphotography for the accurate monitoring of a ...

生物工学

Cryopreservation of Preimplantation Embryos of Cattle, Sheep, and Goats

Preimplantation embryos from cattle, sheep, and goats may be cryopreserved for short- or long-term storage. Preimplantation embryos consist predominantly of water, and the avoidance of intracellular ice crystal formation during the cryopreservation process is of paramount importance to maintain embryo viability. Embryos are placed into a hypertonic solution (1.4 &#x2013; 1.5 M) of a cryoprotective agent (CPA) such as ethylene glycol (EG) or glycerol (GLYC) to create an osmotic gradient that facilitates cellular dehydration. After embryos reach osmotic equilibrium in the CPA solution, they are individually loaded in the hypertonic CPA solution into 0.25 ml plastic straws for freezing. Embryos are placed into a controlled rate freezer at a temperature of -6&deg;C. Ice crystal formation is induced in the CPA solution surrounding the embryo, and crystallization causes an increase in the concentration of CPA outside of the embryo, causing further cellular dehydration. Embryos are cooled at a rate of 0.5&deg;C/min, enabling further dehydration, to a temperature of -34&deg;C before being plunged into liquid nitrogen (-196&deg;C). Cryopreserved embryos must be thawed prior to transfer to a recipient (surrogate) female. Straws containing the embryos are removed from the liquid nitrogen dewar, held in room temperature air for 3 to 5 sec, and placed into a 37&deg;C water bath for 25 to 30 sec. Embryos cryopreserved in GLYC are placed into a 1 M solution of sucrose for 10 min for removal of the CPA before transfer to a recipient (surrogate) female. Embryos cryopreserved in EG, however, may be directly transferred to the uterus of a recipient.

Preimplantation embryos may be cryopreserved after placement into a hypertonic cryoprotective solution to cause cellular dehydration. After equilibration, ice crystal formation is induced in the solution surrounding the embryo. Further dehydration occurs as the embryo is slowly cooled to subzero temperatures before plunging into liquid nitrogen for storage.

着床前の牛の胚、羊、ヤギの凍結保存

Preimplantation embryos from cattle, sheep, and goats may be cryopreserved for short- or long-term storage.  Preimplantation embryos consist predominantly ...

生物学

Cryopreservation of Mouse Embryos by Ethylene Glycol-Based Vitrification

Cryopreservation of mouse embryos is a technological basis that supports biomedical sciences, because many strains of mice have been produced by genetic modifications and the number is consistently increasing year by year. Its technical development started with slow freezing methods in the 1970s1, then followed by vitrification methods developed in the late 1980s2. Generally, the latter technique is advantageous in its quickness, simplicity, and high survivability of recovered embryos. However, the cryoprotectants contained are highly toxic and may affect subsequent embryo development. Therefore, the technique was not applicable to certain strains of mice, even when the solutions are cooled to 4&deg;C to mitigate the toxic effect during embryo handling. At the RIKEN BioResource Center, more than 5000 mouse strains with different genetic backgrounds and phenotypes are maintained3, and therefore we have optimized a vitrification technique with which we can cryopreserve embryos from many different strains of mice, with the benefits of high embryo survival after vitrifying and thawing (or liquefying, more precisely) at the ambient temperature4.
Here, we present a vitrification method for mouse embryos that has been successfully used at our center. The cryopreservation solution contains ethylene glycol instead of DMSO to minimize the toxicity to embryos5. It also contains Ficoll and sucrose for prevention of devitrification and osmotic adjustment, respectively. Embryos can be handled at room temperature and transferred into liquid nitrogen within 5 min. Because the original method was optimized for plastic straws as containers, we have slightly modified the protocol for cryotubes, which are more easily accessible in laboratories and more resistant to physical damages. We also describe the procedure of thawing vitrified embryos in detail because it is a critical step for efficient recovery of live mice. These methodologies would be helpful to researchers and technicians who need preservation of mouse strains for later use in a safe and cost-effective manner.

An ethylene glycol-based vitrification method for mouse embryos is described. It is advantageous to other methods in its simplicity and low embryonic toxicity, and therefore can be broadly applicable to many strains of mice, including inbred and gene-modified mice.

エチレングリコール系ガラス化によるマウス胚の凍結保存

Cryopreservation of mouse embryos is a technological basis that supports biomedical sciences, because many strains of mice have been produced by genetic ...

Bulk Droplet Vitrification for Primary Hepatocyte Preservation

Vitrification is a promising ice-free alternative for classic slow-freezing (at approximately 1 &#176;C/min) cryopreservation of biological samples. Vitrification requires extremely fast cooling rates to achieve transition of water into the glass phase while avoiding injurious ice formation. Although pre-incubation with cryoprotective agents (CPA) can reduce the critical cooling rate of biological samples, high concentrations are generally needed to enable ice-free cryopreservation by vitrification. As a result, vitrification is hampered by CPA toxicity and restricted to small samples that can be cooled fast. It was recently demonstrated that these inherent limitations can be overcome by bulk droplet vitrification. Using this novel method, cells are first pre-incubated with a low intracellular CPA concentration. Leveraging rapid osmotic dehydration, the intracellular CPA is concentrated directly ahead of vitrification, without the need to fully equilibrate toxic CPA concentrations. The cellular dehydration is performed in a fluidic device and integrated with continuous high throughput generation of large sized droplets that are vitrified in liquid nitrogen. This ice-free cryopreservation method with minimal CPA toxicity is suitable for large cell quantities and results in increased hepatocyte viability and metabolic function as compared to classical slow-freezing cryopreservation. This manuscript describes the methods for successful bulk droplet vitrification in detail.

This manuscript describes an ice-free cryopreservation method for large quantities of rat hepatocytes whereby primary cells are pre-incubated with cryoprotective agents at a low concentration and vitrified in large droplets.

原発性肝細胞保存のためのバルク液滴ガラス化

Vitrification is a promising ice-free alternative for classic slow-freezing (at approximately 1 &#176;C/min) cryopreservation of biological samples. ...

Fertility Preservation Through Oocyte Vitrification: Clinical and Laboratory Perspectives

Preserving female fertility is crucial in a multifunctional healthcare system that takes care of patients' future quality of life. Oocyte cryopreservation is recognized by several international scientific societies as the gold standard for fertility preservation in postpubertal women, for both medical and non-medical indications. The main medical indications are oncologic diseases, gynecologic diseases such as severe endometriosis, systemic diseases compromising the ovarian reserve, and genetic conditions involving premature menopause. This paper describes the whole clinical and laboratory work-up of a fertility preservation treatment by outlining recommendations for objective and evidence-based counseling. Furthermore, it focuses on the effectiveness of the procedure and describes the most appropriate strategies to fully exploit the ovarian reserve and maximize the number of oocytes retrieved in the shortest possible time. The evaluation of the ovarian reserve, the definition of an ideal stimulation protocol, as well as oocyte retrieval, denudation, and vitrification procedures have been detailed along with approaches to maximize their efficacy, efficiency, and safety.

Oocyte cryopreservation is recognized by several international scientific societies as the gold standard for fertility preservation in postpubertal women. Appropriate clinical and laboratory strategies ensure maximum efficacy, efficiency, and safety of fertility preservation treatments.

卵母細胞ガラス化による不妊治療の保存:臨床および検査の視点

Preserving female fertility is crucial in a multifunctional healthcare system that takes care of patients' future quality of life. Oocyte cryopreservation ...

医学

<meta charset="utf8"></head><body>ビトリファイドヒト卵巣皮質組織における卵胞生存率評価

Vitrification of Ovarian Cortex Tissue to Achieve a Glassy State of Aggregation

Ovarian tissue cryopreservation (OTC) is an important option for fertility preservation. For patients whose gonadotoxic treatments cannot be postponed or for pre-pubertal girls, it is often the only option for fertility protection. Cryopreservation can be performed either by vitrification or by slow freezing. Slow freezing is currently the standard approach. An increasing number of studies indicate that vitrification can replace slow freezing in the state-of-the-art in vitro fertilization (IVF) laboratories, significantly improving thawing survival rates and simplifying the technical aspects of cryopreservation. A metal grid-based, high-throughput protocol for rapid vitrification of ovarian cortex tissue, suitable for clinical routine, is described. The sterilization of metal grids and liquid nitrogen ensures high quality, meeting good manufacturing practice (GMP) standards. Vitrification was conducted to ensure ultra-rapid cooling rates. Instead of slowly thawing, samples were rapidly warmed. To assess follicular viability, calcein staining was performed both prior to cryopreservation and after rapid warming. The successful application of vitrification and rapid warming using metal grids is reported. No significant differences in follicular viability were observed prior to vitrification and after rapid warming. These results substantiate the high capacity of tissue vitrification for clinical routine applications as a potential substitute for the widely used slow-freezing method.

<meta charset="utf8"></head><body>著者スポットライト:卵巣組織ガラス化とIn vitro卵胞成長による若年女性がん患者の妊孕性保存の推進

A protocol for the vitrification of ovarian tissue, as an alternative cryopreservation method to the widely used slow freezing protocol, is presented.

ガラス質の凝集状態を達成するための卵巣皮質組織のガラス化

Ovarian tissue cryopreservation (OTC) is an important option for fertility preservation. For patients whose gonadotoxic treatments cannot be postponed or ...