本研究は、中国国家自然科学基金会(82171603)、上海市衛生委員会基金会(202140341)、上海市科学技術委員会(23JC1403803)、NHCおよび上海Key Labsのイノベーション推進プログラムSIBPT(RC2023-02)の支援を受けて行われました。

マウスの取り扱いと実験的研究は、Shanghai Institute for Biomedical and Pharmaceutical Technologiesの動物委員会によって承認されたプロトコルに基づいて行われました。安全手順を確保する:化学物質や生物学的材料を取り扱うときは、常に適切な個人用保護具(PPE)を着用してください。
1. マウス胚の取得
注:このセクションでは、マウス胚を取得するプロセスについて説明します。成体雌マウス(6-8週齢、ハイブリッドC57BL/6およびDBA/2)は、20-25°C、湿度40-60%の12時間の明るい場所と12時間の暗い標準環境条件下で、餌と水 を自由に提供して維持しました。
<ol><li>雌マウスに10IUの妊娠中の牝馬の血清ゴナドトロピン(PMSG)を腹腔内に注射し、次に10IUのヒト絨毛性ゴナドトロピン(HCG)を42〜48時間間隔で注射します。.</li>
	<li>各雌マウスを同等の年齢の雄と交配させ、翌朝膣栓を確認します。 注:膣栓は、精液が膣口で固まった後に形成される淡黄色の坐剤の塊です。</li>
	<li>CO2 吸入により、膣栓で雌マウスを安楽死させます。</li>
	<li>腹部を開き、女性器を見つけます。鉗子で卵管、卵巣、脂肪パッドを伸ばします。卵管と卵巣をハサミで切ります。次に、卵管と子宮を切断します(図1)。</li>
	<li>得られた卵管を、M2培地をあらかじめ充填した35mmのシャーレに移します。 注:M2培地は、インキュベーター(37°C、5%CO2)で30分間予熱する必要があります。</li>
	<li>卵管の肥大した膨大部に25Gの針で穴を開けて、積雲-接合子複合体を解放またはつまみます(図2)。</li>
	<li>鉗子を使用して、卵丘-接合子複合体を0.5 mg/mLヒアルロニダーゼを含むM2溶液に移し、卵丘細胞が解離するまで数分間インキュベートします(図3)。</li>
	<li>卵移植ピペットを使用して受精卵を採取し、4 mg/mL BSAを含むM2培地で洗浄して、残留ヒアルロニダーゼ、卵丘細胞、および不純物を除去します。</li>
</ol>2. マウス胚の in vitro 培養
注:このセクションでは、受精卵から胚盤胞までのマウス胚を in vitro で培養するプロセスについて詳しく説明します。KSOM培養マイクロドロップは、温度とガスの平衡を確保するために、24時間前に調製する必要があります。
<ol><li>35 mmシャーレの底に、それぞれ容量20 μLのKSOM培地を12個調製します。微小液滴を3〜5mLの鉱油で覆い(図4)、ペトリ皿を5%CO2 のインキュベーターに37°Cで平衡化します。</li>
	<li>マウス胚をM2培地からKSOM培地に移し、トランスファーピペットを使用して十分に洗浄します。 注:このステップには、37°Cの加熱ステージを備えた実体顕微鏡が必要です。インキュベーターの外で胚を取り扱う時間を最小限に抑えます。</li>
	<li>洗浄した胚を調製したKSOM微小液滴に入れ、5%CO2 で37°Cで4日間インキュベートします。 注:十分な栄養を確保するために、20μLマイクロドロップあたり5〜10個の胚を培養します。</li>
	<li>サイズ(100〜130μm)、内部細胞質量(内部細胞塊は、すべて圧縮された多数の細胞で構成されている)、栄養膜細胞の数(栄養外皮は、凝集して詰まった多数の細胞で構成されている)、および増殖の程度(胚盤胞は胚の80〜100%を占める)に応じて、有利な胚盤胞を選択します。</li>
</ol>3. 子宮内膜細胞の調製
<ol><li>バイオセーフティキャビネット内で、ピペットを使用して1 mLの2%ゼラチンを吸引し、12ウェル培養プレートに分注します。プレートをゆっくりと回転させて、ゼラチンで底が完全に覆われるようにします。プレートを37°Cのインキュベーターに30分間置きます。その後、余分なゼラチンを吸引し、プレートの底を完全に乾かします。</li>
	<li>コーティングプロセスが完了したら、ヒト子宮内膜腺癌細胞(石川)をDMEM/F12( 詳細は材料表 を参照)、1 mMピルビン酸ナトリウム、2 mM L-グルタミン、100 μg/mLストレプトマイシン、および100 IU/mLペニシリンで培養し、ゼラチンコーティングされた12ウェルプレート(2 × 105細胞/ウェル)に播種し、24時間インキュベートします。</li>
</ol>4. 胚の付着
<ol><li>共培養のために前のステップで調製した石川細胞を含む12ウェルプレートの各ウェルに、5〜15個の新鮮な胚盤胞を静かに配置します(図5)。</li>
	<li>48時間のインキュベーション後、胚を顕微鏡で観察し、付着状態を評価します。胚の付着を観察するには、プレートをすばやく3回動かします。付着していない胚は、石川細胞の表面上で浮遊または転がる動きを示します。</li>
	<li>式(1)を使用して胚の着床率を計算します。胚の接着率を使用して、着床効果を表します。 胚着床率=(付着胚/着床胚の総数)×100%(1)</li>
</ol>

石川細胞を用いたIn Vitro共培養モデルにおける胚接着の評価

<ol>
	<li>Hannan, N. J., Paiva, P., Dimitriadis, E., Salamonsen, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+for+study+of+human+embryo+implantation:+choice+of+cell+lines.">Models for study of human embryo implantation: choice of cell lines.</a> Biol Reprod. 82 (2), 235-245 (2010).</li><li>Li, H. 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=Establishment+of+an+efficient+method+to+quantify+embryo+attachment+to+endometrial+epithelial+cell+monolayers.">Establishment of an efficient method to quantify embryo attachment to endometrial epithelial cell monolayers.</a> In Vitro Cell Dev Biol Anim. 38 (9), 505-511 (2002).</li><li>Hohn, H. P., Linke, M., Denker, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion+of+trophoblast+to+uterine+epithelium+as+related+to+the+state+of+trophoblast+differentiation:+in+vitro+studies+using+cell+lines.">Adhesion of trophoblast to uterine epithelium as related to the state of trophoblast differentiation: in vitro studies using cell lines.</a> Mol Reprod Dev. 57 (2), 135-145 (2000).</li><li>Ho, H., Singh, H., Aljofan, M., Nie, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+in+vitro+model+of+human+embryo+attachment.">A high-throughput in vitro model of human embryo attachment.</a> Fertil Steril. 97 (4), 974-978 (2012).</li><li>Karvas, R. M., 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=Stem-cell-derived+trophoblast+organoids+model+human+placental+development+and+susceptibility+to+emerging+pathogens.">Stem-cell-derived trophoblast organoids model human placental development and susceptibility to emerging pathogens.</a> Cell Stem Cell. 29 (5), 810-825 (2022).</li><li>Xiang, L., 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=A+developmental+landscape+of+3D-cultured+human+pre-gastrulation+embryos.">A developmental landscape of 3D-cultured human pre-gastrulation embryos.</a> Nature. 577 (7791), 537-542 (2020).</li><li>Ojosnegros, S., Seriola, A., Godeau, A. L., Veiga, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryo+implantation+in+the+laboratory:+an+update+on+current+techniques.">Embryo implantation in the laboratory: an update on current techniques.</a> Hum Reprod Update. 27 (3), 501-530 (2021).</li><li>Berneau, S. C., 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=Characterisation+of+osteopontin+in+an+in+vitro+model+of+embryo+implantation.">Characterisation of osteopontin in an in vitro model of embryo implantation.</a> Cells. 8 (5), 432 (2019).</li><li>Jiang, R., 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=Enhanced+HOXA10+sumoylation+inhibits+embryo+implantation+in+women+with+recurrent+implantation+failure.">Enhanced HOXA10 sumoylation inhibits embryo implantation in women with recurrent implantation failure.</a> Cell Death Discov. 3, 17057 (2017).</li><li>Kang, Y. J., Forbes, K., Carver, J., Aplin, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+osteopontin-integrin+&#945;v&#946;3+interaction+at+implantation:+functional+analysis+using+three+different+in+vitro+models.">The role of the osteopontin-integrin &#945;v&#946;3 interaction at implantation: functional analysis using three different in vitro models.</a> Hum Reprod. 29 (4), 739-749 (2014).</li><li>Duan, C. C., 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=Oocyte+exposure+to+supraphysiological+estradiol+during+ovarian+stimulation+increased+the+risk+of+adverse+perinatal+outcomes+after+frozen-thawed+embryo+transfer:+a+retrospective+cohort+study.">Oocyte exposure to supraphysiological estradiol during ovarian stimulation increased the risk of adverse perinatal outcomes after frozen-thawed embryo transfer: a retrospective cohort study.</a> J Dev Orig Health Dis. 11 (4), 392-402 (2020).</li><li>Chen, G., 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=Integrins+&#946;1+and+&#946;3+are+biomarkers+of+uterine+condition+for+embryo+transfer.">Integrins &#946;1 and &#946;3 are biomarkers of uterine condition for embryo transfer.</a> J Transl Med. 14 (1), 303-312 (2016).</li><li>Liang, X., 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=Transgelin+2+is+required+for+embryo+implantation+by+promoting+actin+polymerization.">Transgelin 2 is required for embryo implantation by promoting actin polymerization.</a> FASEB J. 33 (4), 5667-5675 (2019).</li><li>Green, C. J., Fraser, S. T., Day, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin-like+growth+factor+1+increases+apical+fibronectin+in+blastocysts+to+increase+blastocyst+attachment+to+endometrial+epithelial+cells+in+vitro.">Insulin-like growth factor 1 increases apical fibronectin in blastocysts to increase blastocyst attachment to endometrial epithelial cells in vitro.</a> Hum Reprod. 30 (2), 284-298 (2015).</li><li>Kang, Y. J., 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=MiR-145+suppresses+embryo-epithelial+juxtacrine+communication+at+implantation+by+modulating+maternal+IGF1R.">MiR-145 suppresses embryo-epithelial juxtacrine communication at implantation by modulating maternal IGF1R.</a> J Cell Sci. 128 (4), 804-814 (2015).</li><li>Berger, C., Boggavarapu, N. R., Menezes, J., Lalitkumar, P. G., Gemzell-Danielsson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+ulipristal+acetate+on+human+embryo+attachment+and+endometrial+cell+gene+expression+in+an+in+vitro+co-culture+system.">Effects of ulipristal acetate on human embryo attachment and endometrial cell gene expression in an in vitro co-culture system.</a> Hum Reprod. 30 (4), 800-811 (2015).</li><li>Li, X., 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=Three-dimensional+culture+models+of+human+endometrium+for+studying+trophoblast-endometrium+interaction+during+implantation.">Three-dimensional culture models of human endometrium for studying trophoblast-endometrium interaction during implantation.</a> Reprod Biol Endocrinol. 20 (1), 120-138 (2022).</li><li>Fitzgerald, H. C., Schust, D. J., Spencer, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+models+of+the+human+endometrium:+evolution+and+application+for+women's+health.">In vitro models of the human endometrium: evolution and application for women's health.</a> Biol Reprod. 104 (2), 282-293 (2021).</li><li>Ahn, J., 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=Three-dimensional+microengineered+vascularised+endometrium-on-a-chip.">Three-dimensional microengineered vascularised endometrium-on-a-chip.</a> Hum Reprod. 36 (10), 2720-2731 (2021).</li><li>Sternberg, A. K., Buck, V. U., Classen-Linke, I., Leube, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+mechanical+forces+change+the+human+endometrium+during+the+menstrual+cycle+in+preparation+for+embryo+implantation.">How mechanical forces change the human endometrium during the menstrual cycle in preparation for embryo implantation.</a> Cells. 10 (8), 2008 (2021).</li></ol>

著者は、宣言すべき金銭的またはその他の利益相反はありません。

提案された胚移植のためのin vitroモデルのシンプルさと迅速さは、初期段階の薬物スクリーニングやその他の研究アプリケーションに顕著な利点を提供します。簡便なプロトコールと石川細胞株のハイスループットな性質により、特に創薬の初期段階における大規模なスクリーニングの理想的な候補となります。Liang13は、TAGLN2のノックダウンが栄養膜細胞の浸潤能力?...

妊娠を成功させるための最初のステップである胚移植は、その生物学的基盤を理解し、不妊症の課題に対処するために重要です。しかし、倫理的な制約により、ヒト臨床胚サンプルの収集には大きな制限があり、妊娠初期におけるヒト胚と子宮内膜との間の複雑な相互作用に関する研究が妨げられています1。これらの複雑なメカニズムを深く理解することは、基礎生物学研究、創薬、およびリプロダクティブヘルスを前進させるために不可欠です。
以前のin vitroモデルでは、単層ヒト子宮内膜上皮細胞(RL95-2)2 をBeWo、JAr、Jeg-33 絨毛がん細胞などの胚性代替細胞と共培養した接着モデルが採用されていました。それにもかかわらず、細胞凝集体が大きいか小さいかによって実験の精度が損なわれる可能性があるため、70〜100μmの範囲のスフェロイドのサイズを選択することは重要でした。特に、各調製物中のスフェロイドのうち、適切なサイズ4の基準を満たしたのはわずか25%であり、胚盤胞の生理学的条件と比較して有意な差が見られました。
さらに、子宮内膜および胚盤胞の3次元(3D)培養システムは、より生理学的に関連性のある環境を提供します5が、高い技術的専門知識、細胞増殖の遅延、高いリソースコストとメンテナンスコスト、標準化の難しさ、および低い再現性6を必要とします。
このプロトコルでは、ほとんどの研究室で開発および利用できる、費用対効果が高く、迅速な胚着床のin vitroモデルを提示します。この方法の全体的な目的は、制御された環境で胚と子宮内膜との間の相互作用を研究するための信頼性と再現性のあるプラットフォームを提供することです。このモデルは、 in vitroでの胚着床の主要なイベントをシミュレートすることにより、動物モデルと臨床環境との間のギャップを埋め、より正確で的を絞った研究を可能にすることを目的としています。
胚代替物と比較して、マウス胚盤胞の使用は、in vivo胚着床プロセスのより生理学的に関連性のある表現を提供します7。また、ヒト子宮内膜腺がん由来の石川細胞株は、腺上皮と子宮内腔1の混合特性を有しており、正常な子宮内膜と同様の酵素、構造タンパク質、機能的ステロイド受容体を発現することが可能になり、胚着床のための生理学的に重要な基質を提供します。共培養システムのシンプルさと迅速性により、ハイスループットスクリーニングと薬物試験が可能になります8,9,10。堅牢で再現性のあるプラットフォームを提供することにより、この方法は、胚の着床とそれが人間の健康に与える影響についての理解を深める機会を提供します。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>17-&#946;-estradiol</td>
			<td>SIGMA</td>
			<td>3301</td>
			<td>Most potent mammalian estrogenic hormone</td>
		</tr>
		<tr>
			<td>BD Falcon</td>
			<td>BD</td>
			<td>353001</td>
			<td>Bacteriological Petri Dishes 35 x 10 mm style w/tight lid, crystal-grade virgin polystyrene, sterile</td>
		</tr>
		<tr>
			<td>Biosafety Cabinet</td>
			<td>ESCO</td>
			<td>class <img alt="Equation 1" src="/files/ftp_upload/66873/66873eq01.jpg" style="margin: auto;" />BSC</td>
			<td>Aseptic operations, making culture dishes, aliquoting reagents, etc</td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Thermo</td>
			<td>8000DH</td>
			<td>Embryo culture</td>
		</tr>
		<tr>
			<td>Culture plate</td>
			<td>Corning</td>
			<td>3506</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>1133032</td>
			<td>DMEM/F12 (1x), liquid 1:1,Contains L-glutamine and 15 mM HEPES buffer</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10099141</td>
			<td>Fetal Bovine Serum, Qualified, Australia Origin</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>SIGMA</td>
			<td>G9391</td>
			<td>Type B, powder, BioReagent, suitable for cell culture</td>
		</tr>
		<tr>
			<td>HCG</td>
			<td>Nanjing Aibei</td>
			<td>M2520</td>
			<td>Sterilization reagent, intraperitoneal injection, 50 IU/mL</td>
		</tr>
		<tr>
			<td>Hyaluronidase&#160;</td>
			<td>SIGMA</td>
			<td>V900833</td>
			<td>Reagent grade, powder&#160;</td>
		</tr>
		<tr>
			<td>KSOM</td>
			<td>Merck</td>
			<td>MR-020P-D</td>
			<td>(1x), Powder, w/o Phenol Red, 5 x 10 mL</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>25030081</td>
			<td>L-glutamine-200 mM (100x), liquid</td>
		</tr>
		<tr>
			<td>M2</td>
			<td>Merck</td>
			<td>MR-015-D</td>
			<td>EmbryoMax M2 Medium (1x), Liquid, with Phenol Red</td>
		</tr>
		<tr>
			<td>Mineral oil&#160;</td>
			<td>SIGMA</td>
			<td>M8410</td>
			<td>Mineral oil is suitable for use as a cover layer to control evaporation and cross-contamination in various molecular biology applications</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin, liquid</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>10,000 Units penicillin (base) and 10,000 units streptomycin (base), utilizing penicillin G (sodium salt) and streptomycin sulfate in 0.85% saline</td>
		</tr>
		<tr>
			<td>PMSG</td>
			<td>Nanjing Aibei</td>
			<td>M2620</td>
			<td>Sterilization reagent ,intraperitoneal injection, 50 IU/mL</td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>SIGMA</td>
			<td>5341</td>
			<td>Steroid hormone secreted by the corpus luteum during the latter half of the menstrual cycle</td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Gibco</td>
			<td>11360070</td>
			<td>Sodium pyruvate is commonly added to cell culture media as a carbon source in addition to glucose</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Olympus</td>
			<td>SZX7</td>
			<td>Embryo retrieval and observation of embryo development</td>
		</tr>
	</tbody>
</table>

establishment of an embryo implantation model in vitro

胚の着床は、妊娠を成功させるための最初のステップです。胚着床のための in vitro モデルは、基礎生物学研究、医薬品開発、スクリーニングに不可欠です。この論文では、胚移植のためのシンプルで迅速、かつ高効率な in vitro モデルを紹介します。本プロトコールでは、まずマウス胚盤胞の獲得とヒト子宮内膜腺癌細胞(石川)の移植用調製物を導入し、続いてマウス胚と石川細胞の共培養法を導入します。最後に、このモデルに基づいて、17-β-エストラジオール(E2)とプロゲステロン(P4)のさまざまな濃度が胚の接着率に与える影響を評価する研究を実施しました。私たちの調査結果は、高濃度のE2 が胚の接着を有意に減少させるのに対し、プロゲステロンの添加は接着率を回復させることを明らかにしました。このモデルは、サイトカイン、薬物、着床と子宮内膜の受容性を制御する転写因子など、接着プロセスに関与する分子を評価およびスクリーニングするためのシンプルで迅速なプラットフォームを提供します。

生殖補助医療の過程では、制御された卵巣過剰刺激(COH)が重要なステップであり、患者のエストロゲンレベルが自然周期と比較して10〜20倍以上大幅に上昇します11。このような状況を踏まえて、高血清エストロゲン濃度が新鮮胚移植時の胚着床に影響を与えるかどうかを尋ねた。この胚着床モデルに基づいて、我々は、胚着床率12に対するE2およびP4...

著者スポットライト:胚と子宮内膜の相互作用を分析することにより、妊娠率を改善するための治療戦略の推進

このプロトコルは、胚移植プロセスに影響を与える関連分子を評価するためのシンプルで効率的な胚移植 in vitro モデルを確立するための方法論を説明しています。

In Vitroでの胚着床モデルの確立

Embryo implantation is the first step in the establishment of a successful pregnancy. An in vitro model for embryo implantation is critical for basic ...

establishment-of-an-embryo-implantation-model-in-vitro

Research

JoVE Journal

Biology

Establishment of an Embryo Implantation Model In Vitro

414 ビュー.  Fudan University. 胚移植は、妊娠を成功させるための最初のステップであり、その生物学的基盤を理解し、不妊症の課題に対処するために重要です。この研究の目的は、制御された条件下で胚の子宮内膜の相互作用を分析するための一貫したプラットフォームを確立することです。このin vitroモデルに基づいて、超高濃度のエストロゲンは胚着床リスクを大幅に?...

ビデオ: 著者スポットライト:胚と子宮内膜の相互作用を分析することにより、妊娠率を改善するための治療戦略の推進

Christopher Hughes:  An in vitro model for the Study of Angiogenesis (Interview)

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

Christopher Hughes:  An in vitro model for the Study of Angiogenesis Interview

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro. He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

クリストファーヒューズ：血管新生の研究のためのin vitroモデル（インタビュー）

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that ...

生物学

An In Vitro Skin Irritation Test (SIT) using the EpiDerm Reconstructed Human Epidermal (RHE) Model

The EpiDerm Skin Irritation test (EpiDerm SIT) was developed (1,2,3) and validated (4,5) for in vitro skin irritation testing of chemicals, including cosmetic and pharmaceutical ingredients. The EpiDerm SIT utilizes the 3D in vitro reconstructed human epidermal (RHE) model EpiDerm. The procedure described in this protocol allows for discrimination between irritants of GHS category 2 and non-irritants (6). The test is performed over the course of a 4 day time period, consisting of pre-incubation, 60 minute exposure, 42 hour post-incubation and MTT viability assay. After tissue receipt and overnight pre-incubation (Day 0), tissues are topically exposed to the test chemicals (Day 1), which can be liquid, semi-solids, solid or wax. Three tissues are used for each test chemical, as well as for the positive control (5% aq. SDS solution), and a negative control (DPBS). Chemical exposure lasts for 60 minutes, 35 min of which the tissues are kept in an incubator at 37&deg;C. The test substances are then removed from the tissue surface by an extensive washing procedure. The tissue inserts are blotted and transferred to fresh medium. After a 24 hr incubation period (Day 2), the medium is exchanged. The medium can be saved for further analysis of cytokines or other endpoints of interest. After the medium exchange, tissues are incubated for an additional 18 hours. At the end of the entire 42h post-incubation (day 3), the tissues are transferred into yellow MTT solution and incubated for 3 hours. The resultant purple-blue formazan salt, formed mainly by mitochondrial metabolism, is extracted for 2 hours using isopropanol. The optical density of the extracted formazan is determined using a spectrophotometer. A chemical is classified as an irritant if the tissue viability relative to the negative control treated tissues is reduced below 50%. This procedure can be used as full replacement of the in vivo rabbit skin irritation test for hazard identification and labeling of chemicals in line with EU regulations (7).

An In Vitro Skin Irritation Test SIT using the EpiDerm Reconstructed Human Epidermal RHE Model

In this video, we demonstrate the EpiDerm Skin Irritation test (EpiDerm SIT) developed and validated for in vitro skin irritation testing of chemicals, including cosmetic and pharmaceutical ingredients.

アンインビトロ</em表皮を使用して>皮膚刺激性試験（SIT）は、ヒト上皮（RHE）モデルを再構築

The EpiDerm Skin Irritation test (EpiDerm SIT) was developed (1,2,3) and validated (4,5) for in vitro skin irritation testing of chemicals, including ...

An In vitro FluoroBlok Tumor Invasion Assay

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to both secrete proteases that break down the basement membrane as well as migrate in order to be invasive. BD BioCoat Tumor Invasion System provides cells with conditions that allow assessment of their invasive property in vitro1,2. It consists of a BD Falcon FluoroBlok 24-Multiwell Insert Plate with an 8.0 micron pore size PET membrane that has been uniformly coated with BD Matrigel Matrix. This uniform layer of BD Matrigel Matrix serves as a reconstituted basement membrane in vitro providing a true barrier to non-invasive cells while presenting an appropriate protein structure to study invasion. The coating process occludes the pores of the membrane, blocking non-invasive cells from migrating through the membrane. In contrast, invasive cells are able to detach themselves from and migrate through the coated membrane. Quantitation of cell invasion can be achieved by either pre- or post-cell invasion labeling with a fluorescent dye such as DiIC12(3) or calcein AM, respectively, and measuring the fluorescence of invading cells. Since the BD FluoroBlok membrane effectively blocks the passage of light from 490-700 nm at &gt;99% efficiency, fluorescently-labeled cells that have not invaded are not detected by a bottom-reading fluorescence plate reader. However, cells that have invaded to the underside of the membrane are no longer shielded from the light source and are detected with the respective plate reader. This video demonstrates an endpoint cell invasion assay, using calcein AM to detect invaded cells.

An In vitro FluoroBlok Tumor Invasion Assay

This video demonstrates how to measure cell invasion through cell culture inserts using a fluorescence-based methodology.

アン試験管内でフルオロブロックの腫瘍の浸潤アッセイ

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to ...

Utero-tubal Embryo Transfer and Vasectomy in the Mouse Model

The transfer of preimplantation embryos to a surrogate female is a required step for the production of genetically modified mice or to study the effects of epigenetic alterations originated during preimplantation development on subsequent fetal development and adult health. The use of an effective and consistent embryo transfer technique is crucial to enhance the generation of genetically modified animals and to determine the effect of different treatments on implantation rates and survival to term. Embryos at the blastocyst stage are usually transferred by uterine transfer, performing a puncture in the uterine wall to introduce the embryo manipulation pipette. The orifice performed in the uterus does not close after the pipette has been withdrawn, and the embryos can outflow to the abdominal cavity due to the positive pressure of the uterus. The puncture can also produce a hemorrhage that impairs implantation, blocks the transfer pipette and may affect embryo development, especially when embryos without zona are transferred. Consequently, this technique often results in very variable and overall low embryo survival rates. Avoiding these negative effects, utero-tubal embryo transfer take advantage of the utero-tubal junction as a natural barrier that impedes embryo outflow and avoid the puncture of the uterine wall. Vasectomized males are required for obtaining pseudopregnant recipients. A technique to perform vasectomy is described as a complement to the utero-tubal embryo transfer.

Utero-tubal embryo transfer uses the utero-tubal junction as a barrier to prevent the embryo outflow that may occur when performing uterine transfer. Vasectomized males are required to obtain pseudopregnant recipients for embryo transfer. Both techniques are discussed.

マウスモデルにおける子宮卵管胚移植と精管切除

The transfer of preimplantation embryos to a surrogate female is a required step for the production of genetically modified mice or to study the effects ...

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and develops most often in the setting of immunosuppression. Despite decades of research, optimal treatment of KS remains poorly defined and clinical outcomes are especially unfavorable in resource-limited settings. KS lesions are driven by pathological angiogenesis, chronic inflammation, and oncogenesis, and various in vitro cell culture models have been developed to study these processes. KS arises from KSHV-infected cells of endothelial origin, so EC-lineage cells provide the most appropriate in vitro surrogates of the spindle cell precursor. However, because EC have a limited in vitro lifespan, and as the oncogenic mechanisms employed by KSHV are less efficient than those of other tumorigenic viruses, it has been difficult to assess the processes of transformation in primary or telomerase-immortalized EC. Therefore, a novel EC-based culture model was developed that readily supports transformation following infection with KSHV. Ectopic expression of the E6 and E7 genes of human papillomavirus type 16 allows for extended culture of age- and passage-matched mock- and KSHV-infected EC and supports the development of a truly transformed (i.e., tumorigenic) phenotype in infected cell cultures. This tractable and highly reproducible model of KS has facilitated the discovery of several essential signaling pathways with high potential for translation into clinical settings.

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is a tumor induced by infection with the oncogenic virus human herpesvirus-8/KS herpesvirus (HHV-8/KSHV). The endothelial cell culture model described here is uniquely suited for studying the mechanisms by which KSHV transforms host cells.

カポジ肉腫ヘルペス ウイルスによる細胞変換を勉強するための in Vitroモデル

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and ...

Establishment of an Extracellular Acidic pH Culture System

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the role of acidic extracellular pH in tumor aggressiveness and its underlying mechanism has not been extensively studied compared to hypoxic or nutrient starvation conditions. In addition, a well-defined culture method to mimic the acidic extracellular tumor microenvironment has not been fully reported.
Here we present a simple in vitro culture method to maintain acidic extracellular pH using reduced bicarbonate and increased lactate or HCl concentrations in the culture medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h following culture of PANC-1 and AsPC-1 pancreatic cancer cells. Three distinct acidic media conditions in this study highly upregulated pH-responsive genes such as MSMO1, INSIG1, and IDI1 compared to hypoxia or nutrient starvation. The upregulation of these genes can be used as a marker of acidic pH. These simple techniques are beneficial to elucidate underlying mechanisms of tumor malignancy under acidic tumor microenvironment. Therefore, our extracellular acidic pH culture system enables discovery of cellular acidic pH responses not only in cancer cells but also in primary cells, such as renal tubular cells, in relation to the other acidic disorders including, diabetic ketoacidosis, lactic acidosis, renal tubular acidosis, and respiratory acidosis.

The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems.

酸性細胞外 pH 培養系の確立

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...

Implantation of Electrospun Vascular Grafts with Optimized Structure in a Rat Model

Here, we present a protocol to fabricate macroporous PCL vascular graft and describe an evaluation protocol by using a rat model of abdominal aorta replacement. The electrospun vascular grafts often possess relatively small pores, which limit cell infiltration into the grafts and hinder the regeneration and remodeling of the neo-arteries. In this study, PCL vascular grafts with thicker fibers (5 - 6 &#181;m) and larger pores (~30 &#181;m) were fabricated by using a modified processing technique. The long-term performance of the graft was evaluated by implantation in a rat abdominal aorta model. Ultrasound analysis showed that the grafts remained patent without aneurysm or stenosis occurring even after 12 months of implantation. Macroporous structure improved the cell ingrowth and thus promoted tissue regenerated at 3 months. More importantly, there was no sign of adverse remodeling, such as calcification within the graft wall after 12 months. Therefore, electrospun PCL vascular grafts with modified macroporous processing hold potential to be an artery substitute for long-term implantation.

Here, we present a modified electrospinning method to fabricate PCL vascular grafts with thick fibers and large pores, and describe a protocol to evaluate the in vivo performance in a rat model of abdominal aorta replacement.

エレクトロスピニング ラットモデルにおける最適化された構造を有する人工血管の移植

Here, we present a protocol to fabricate macroporous PCL vascular graft and describe an evaluation protocol by using a rat model of abdominal aorta ...

An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm

Staphylococcus aureus (S. aureus) is a common human pathogen, which causes pyogenic and systemic infections. S. aureus infections are difficult to eradicate not only due to the emergence of antibiotic-resistant strains but also its ability to form biofilms. Recently, photodynamic therapy (PDT) has been indicated as one of the potential treatments for controlling biofilm infections. However, further studies are required to improve our knowledge of its effect on bacterial biofilms, as well as the underlying mechanisms. This manuscript describes an in vitro model of PDT with 5-aminolevulinic acid (5-ALA), a precursor of the actual photosensitizer, protoporphyrin IX (PpIX). Briefly, mature S. aureus biofilms were incubated with ALA and then exposed to light. Subsequently, the antibacterial effect of ALA-PDT on S. aureus biofilm was quantified by calculating the colony forming units (CFUs) and visualized by viability fluorescent staining via confocal laser scanning microscopy (CLSM). Representative results demonstrated a strong antibacterial effect of ALA-PDT on S. aureus biofilms. This protocol is simple and can be used to develop an in vitro model to study the treatment of S. aureus biofilms with ALA-PDT. In the future, it could also be referenced in PDT studies utilizing other photosensitizers for different bacterial strains with minimal adjustments.

An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm

This manuscript describes a protocol to study the antimicrobial effect of 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) on a Staphylococcus aureus biofilm. This protocol can be used to develop an in vitro model to study the treatment of bacterial biofilms with PDT in the future.

黄色ブドウ球菌のバイオ フィルムに及ぼす 5-アミノレブリン酸による光線力学療法を勉強する体外モデル

Staphylococcus aureus (S. aureus) is a common human pathogen, which causes pyogenic and systemic infections. S. aureus infections are difficult to ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

EYFP タグ付き CENP-A (EYFP-CENP-A) のユビキタス性を同定するための質量分析分析

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

The Establishment of a Murine Mandibular Molar Extraction Socket Healing Model

This study introduces the development of a molar extraction model in the murine mandible to provide a practicable model for studying alveolar bone regeneration and intramembranous ossification. C57/J6 mice were used to extract the mandibular first molar to establish this model. They were euthanized, and the bilateral mandibles harvested, at 1 week and 4 weeks post-surgery, respectively. Subsequent serial stereoscopic harvest, histological assessment, and immunofluorescence staining were performed to demonstrate successful surgery. Immediately after surgery, the stereoscopic images displayed an empty extraction socket. The hematoxylin and eosin (H&#38;E) at 1 week and Masson staining at 4 weeks post-surgery showed that the area of the original root was partially and fully filled with bone trabeculae, respectively. The immunofluorescence staining showed that, compared with the homeostasis side, the Sp7 expression increased at 1 week post-surgery, suggesting vigorous osteogenesis in the alveolar fossa. All these results demonstrated a practicable murine tooth extraction socket healing model. Upcoming studies revealing the mechanisms of jawbone defect healing or socket healing could adopt this method.

This protocol demonstrates step-by-step details of how to extract the mandibular first molar in the mouse. It provides an alternative method for researchers focusing on jawbone healing and regeneration.