Çalışmalarımız Çin Ulusal Doğa Bilimleri Vakfı (82171603), Şanghay Belediye Sağlık Komisyonu Vakfı (202140341), Şanghay Belediyesi Bilim ve Teknoloji Komisyonu (23JC1403803) ve NHC ve Shanghai Key Labs, SIBPT İnovasyon Teşvik Programı (RC2023-02) tarafından desteklenmiştir.

Fare işleme ve deneysel çalışmalar, Şanghay Biyomedikal ve Farmasötik Teknolojiler Enstitüsü Hayvan Komitesi tarafından onaylanan protokoller altında gerçekleştirildi. Güvenlik Prosedürlerini Sağlayın: Kimyasalları veya biyolojik malzemeleri kullanırken daima uygun kişisel koruyucu ekipman (KKD) kullanın.
1. Fare embriyolarının elde edilmesi
NOT: Bu bölümde fare embriyolarının elde edilme süreci açıklanmaktadır. Yetişkin dişi fareler (6-8 haftalık, hibrit C57BL / 6 ve DBA / 2), 20-25 ° C'de ve% 40-60 nemde 12 saat aydınlık ve 12 saat karanlık standart çevre koşulları altında tutuldu.
<ol><li>Dişi farelere intraperitoneal olarak 10 IU hamile kısrak serum gonadotropini (PMSG) enjekte edin ve daha sonra 42-48 saat aralıklarla 10 IU insan koryonik gonadotropin (HCG) enjekte edin.</li>
	<li>Her dişi fareyi benzer yaştaki bir erkekle eşleştirin ve ertesi sabah vajinal tıkaç olup olmadığını kontrol edin. NOT: Vajinal tıkaç, meni vajinal açıklıkta katılaştıktan sonra oluşan açık sarı bir fitil yumrudur.</li>
	<li>Dişi fareleri CO2 inhalasyonu ile vajinal tıkaçlarla ötenazi yapın.</li>
	<li>Karnı açın ve kadın cinsel organını bulun; yumurta kanalı, yumurtalık ve yağ yastıkçıklarını forseps ile gerin; yumurta kanalını ve yumurtalığı makasla kesin; ve sonra yumurta kanalını ve uterusu kesin (Şekil 1).</li>
	<li>Elde edilen yumurta kanalını M2 ortamı ile önceden doldurulmuş 35 mm'lik bir Petri kabına aktarın. NOT: M2 ortamı bir inkübatörde (37 °C, %5 CO2) 30 dakika önceden ısıtılmalıdır.</li>
	<li>Kümülüs-zigot kompleksini serbest bırakmak veya sıkıştırmak için yumurta kanalının genişlemiş ampullasını 25 G'lik bir iğne ile delin (Şekil 2).</li>
	<li>Kümülüs-zigot kompleksini 0.5 mg / mL hyaluronidaz içeren M2 çözeltisine aktarmak için forseps kullanın ve kümülüs hücreleri ayrışana kadar birkaç dakika inkübe edin (Şekil 3).</li>
	<li>Zigotları bir yumurta transfer pipeti kullanarak toplayın ve kalıntı hyaluronidaz, kümülüs hücreleri ve safsızlıkları gidermek için 2 mg / mL BSA içeren M4 ortamında yıkayın.</li>
</ol>2. Fare embriyosunun in vitro kültürü
NOT: Bu bölüm, fare embriyolarının zigotlardan blastosistlere kadar in vitro kültürleme sürecini detaylandırmaktadır. Sıcaklık ve gaz dengesini sağlamak için KSOM kültürü mikro damlaları 24 saat önceden hazırlanmalıdır.
<ol><li>35 mm'lik bir Petri kabının dibinde, her biri 20 μL hacme sahip 12 mikro damlacık KSOM ortamı hazırlayın. Mikro damlacıkları 3-5 mL mineral yağ ile örtün (Şekil 4) ve Petri kaplarını dengeleme için %5 CO2 ile 37 °C'de bir inkübatöre yerleştirin.</li>
	<li>Fare embriyolarını kapsamlı yıkama için bir transfer pipeti kullanarak M2 ortamından KSOM ortamına aktarın. NOT: Bu adım, 37 °C ısıtma aşamasına sahip bir stereomikroskop gerektirir. Kuluçka makinesinin dışındaki embriyo işleme süresini en aza indirin.</li>
	<li>Yıkanmış embriyoları hazırlanan KSOM mikro damlacıklarına yerleştirin ve 4 gün boyunca% 5 CO2 ile 37 ° C'de inkübe edin. NOT: Yeterli beslenmeyi sağlamak için, 20 μL mikro damla başına 5-10 embriyo kültürleyin.</li>
	<li>Avantajlı blastokistleri büyüklüklerine (100-130 μm), iç hücre kütlesine (iç hücre kütlesi hepsi sıkıştırılmış birçok hücreden oluşur), trofoblast hücrelerinin sayısına (trofektodermis birbirine bağlı ve birlikte paketlenmiş birçok hücreden oluşur) ve genişleme derecesine (blastocoel embriyonun %80-100'ünü kaplar) göre seçin.</li>
</ol>3. Endometrial hücrelerin hazırlanması
<ol><li>Bir biyogüvenlik kabininin içinde, bir pipet kullanarak 1 mL %2 jelatini aspire edin ve 12 oyuklu bir kültür plakasına dağıtın. Alt kısmın jelatin ile tamamen kaplanmasını sağlamak için plakayı yavaşça döndürün. Plakayı 37 °C'lik bir inkübatörde 30 dakika boyunca yerleştirin. Daha sonra fazla jelatini aspire edin ve plakanın tabanının tamamen kurumasını bekleyin.</li>
	<li>Kaplama işlemi tamamlandıktan sonra, insan endometriyal adenokarsinom hücrelerini (Ishikawa) DMEM/F12 (ayrıntılar için Malzeme Tablosuna bakınız), 1 mM sodyum piruvat, 2 mM L-glutamin, 100 μg/mL streptomisin ve 100 IU/mL penisilin ile kültürleyin, bunları jelatin kaplı 12 oyuklu plakalara (2 × 105hücre/oyuk) tohumlayın ve 24 saat inkübe edin.</li>
</ol>4. Embriyo bağlantısı
<ol><li>Ko-kültür için önceki adımda hazırlanan Ishikawa hücrelerini içeren 12 oyuklu plakanın her bir oyuğuna 5-15 taze blastosisti nazikçe yerleştirin (Şekil 5).</li>
	<li>48 saatlik inkübasyondan sonra, bağlanma durumlarını değerlendirmek için embriyoları mikroskop altında gözlemleyin. Embriyo tutunmasını gözlemlemek için, plakayı üç kez hızlı bir şekilde hareket ettirin. Bağlanmamış embriyolar, Ishikawa hücrelerinin yüzeyi üzerinde yüzer veya yuvarlanma hareketi sergileyecektir.</li>
	<li>Denklem (1) kullanarak embriyo implantasyon oranını hesaplayın; İmplantasyon etkisini temsil etmek için embriyo yapışma oranını kullanın. Embriyo İmplantasyon Oranı = (Bağlı Embriyo / Toplam İmplante Edilen Embriyo Sayısı) × %100 (1)</li>
</ol>

Ishikawa Hücreleri ile İn Vitro Ko-Kültür Modelinde Embriyo Adezyonun Değerlendirilmesi

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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>

Yazarların beyan etmeleri gereken herhangi bir mali veya diğer çıkar çatışmaları yoktur.

Embriyo implantasyonu için önerilen in vitro modelin basitliği ve hızlılığı, erken evre ilaç taraması ve diğer araştırma uygulamaları için dikkate değer avantajlar sunmaktadır. Basit protokol, Ishikawa hücre hatlarının yüksek verimli doğası ile birleştiğinde, özellikle ilaç keşfinin erken aşamalarında büyük ölçekli ekranlar için ideal bir aday haline getirir. Liang13, TAGLN2 yıkımının trofoblast hücrelerinin istila kabiliyetini azalttığını buldu...

Başarılı bir gebeliğin ilk adımı olan embriyo implantasyonu, biyolojik temelini anlamak ve kısırlığın zorluklarını ele almak için çok önemlidir. Bununla birlikte, etik kısıtlamalar, insan klinik embriyo örneklerinin toplanmasında önemli sınırlamalar getirmekte ve hamileliğin erken döneminde insan embriyoları ile endometriyum arasındaki karmaşık etkileşimlere ilişkin araştırmaları engellemektedir1. Bu karmaşık mekanizmaların derinlemesine anlaşılması, temel biyolojik araştırmaları, ilaç keşfini ve üreme sağlığını ilerletmek için hayati önem taşır.
Önceki in vitro modeller, tek katmanlı insan endometriyal epitel hücrelerinin (RL95-2)2 BeWo, JAr veya Jeg-33 koryokarsinom hücreleri gibi embriyonik ikamelerle birlikte kültürlendiği adezyon modellerini kullandı. Bununla birlikte, 70-100 μm arasında değişen sferoidlerin boyutunun seçimi, daha büyük veya daha küçük hücre agregatları deneyin doğruluğunu tehlikeye atabileceğinden çok önemliydi. Özellikle, her preparattaki sferoidlerin sadece% 25'i uygunboyut 4 standardını karşıladı ve blastosistlerin fizyolojik koşullarına kıyasla önemli farklılıklar vardı.
Ayrıca, endometriyum ve blastosist için üç boyutlu (3D) kültür sistemleri fizyolojik olarak daha ilgili bir ortam sunar5, ancak yüksek teknik uzmanlık, yavaş hücre büyümesi, yüksek kaynak ve bakım maliyetleri, standardizasyon zorluğu ve düşük tekrarlanabilirlikgerektirir 6.
Bu protokolde, çoğu laboratuvarda geliştirilebilecek ve kullanılabilecek uygun maliyetli ve hızlı bir in vitro embriyo implantasyon modeli sunuyoruz. Bu yöntemin genel amacı, kontrollü bir ortamda embriyolar ve endometriyum arasındaki etkileşimleri incelemek için güvenilir ve tekrarlanabilir bir platform sağlamaktır. Bu model, in vitro embriyo implantasyonunun temel olaylarını simüle ederek, hayvan modelleri ile klinik ortamlar arasındaki boşluğu doldurmayı ve daha kesin ve hedefli araştırmaları mümkün kılmayı amaçlamaktadır.
Embriyonik ikamelerle karşılaştırıldığında, fare blastosistlerinin kullanımı, in vivo embriyo implantasyon sürecinin fizyolojik olarak daha ilgili bir temsilini sunar7. Ek olarak, insan endometriyal adenokarsinomundan türetilen Ishikawa hücre hattı, glandüler epitel ve uterus lümen epitel1'in karışık özelliklerine sahiptir, bu da normal endometriyumda bulunanlara benzer enzimleri, yapısal proteinleri ve fonksiyonel steroid reseptörlerini eksprese etmesini sağlar, böylece embriyo implantasyonu için fizyolojik olarak ilgili bir substrat sağlar. Ko-kültür sisteminin basitliği ve hızlılığı, yüksek verimli tarama ve uyuşturucu testi yapılmasını mümkün kılar 8,9,10. Bu yöntem, sağlam ve tekrarlanabilir bir platform sağlayarak, embriyo implantasyonu ve bunun insan sağlığı üzerindeki etkisi hakkındaki anlayışımızı ilerletmek için bir fırsat sunar.

<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

Embriyo implantasyonu, başarılı bir gebeliğin kurulmasında ilk adımdır. Embriyo implantasyonu için bir in vitro model, temel biyolojik araştırma, ilaç geliştirme ve tarama için kritik öneme sahiptir. Bu makale, embriyo implantasyonu için basit, hızlı ve oldukça verimli bir in vitro model sunmaktadır. Bu protokolde ilk olarak fare blastokisti edinimi ve insan endometriyal adenokarsinom hücrelerinin (Ishikawa) implantasyon için hazırlığı tanıtıldı, ardından fare embriyoları ve Ishikawa hücreleri için ko-kültür yöntemi uygulandı. Son olarak, bu modele dayalı olarak değişen 17-β-estradiol (E2) ve progesteron (P4) konsantrasyonlarının embriyo adezyon oranları üzerindeki etkisini değerlendirmek için bir çalışma yaptık. Bulgularımız, yüksekE2 konsantrasyonlarının embriyo adezyonunu önemli ölçüde azalttığını, progesteron ilavesinin ise adezyon oranını geri getirebileceğini ortaya koydu. Bu model, implantasyonu ve endometriyal alıcılığı kontrol eden sitokinler, ilaçlar ve transkripsiyon faktörleri gibi adezyon sürecinde yer alan molekülleri değerlendirmek ve taramak için basit ve hızlı bir platform sunar.

Yardımcı üreme teknikleri sürecinde, kontrollü yumurtalık hiperstimülasyonu (COH) çok önemli bir adımdır ve hastalarda östrojen seviyelerinin doğal döngülere kıyasla 10-20 kattan fazla önemli ölçüde artmasına neden olur11. Bu bağlamda, yüksek serum östrojen konsantrasyonlarının taze embriyo transferi sırasında embriyo implantasyonunu etkileyip etkilemediğini sorduk. Bu embriyo implantasyon modeline dayanarak,çeşitli E2 ve P4 konsantrasyonlarının embriyo ...

Yazar Spotlight: Embriyo-Endometriyum Etkileşimlerini Analiz Ederek Gebelik Oranlarını İyileştirmek için Terapötik Stratejilerin Geliştirilmesi

Bu protokol, embriyo implantasyon sürecini etkileyen ilgili molekülleri değerlendirmek için in vitro model olarak basit ve verimli embriyo implantasyonu oluşturmak için metodolojileri açıklar.

İn Vitro Embriyo İmplantasyon Modelinin Kurulması

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 Görüntüleme.  Fudan University. Başarılı bir gebeliğin ilk adımı olan embriyo implantasyonu, biyolojik temelini anlamak ve kısırlığın zorluklarını ele almak için çok önemlidir. Bu çalışmanın amacı, kontrollü koşullar altında embriyo endometrium etkileşimlerini analiz etmek için tutarlı bir platform oluşturmaktır. Bu in vitro modele dayanarak, ultra yüksek östrojen konsantrasyonlarının embriyo implantasyon riskini önemli ölçüde azalttığını, progestero...

Video: Yazar Spotlight: Embriyo-Endometriyum Etkileşimlerini Analiz Ederek Gebelik Oranlarını İyileştirmek için Terapötik Stratejilerin Geliştirilmesi

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.

Christopher Hughes: Bir Anjiyogenez Çalışma in vitro model (Röportaj)

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

Biyoloji

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.

Bir In Vitro</emEpiderm> Cilt tahriş Testi (SIT) İnsan Epidermal (RHE) Model yeniden inşa

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.

Bir In vitro FluoroBlok Tümör Invasion Testi

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.

Fare Modelinde utero-tubal Embriyo Transferi ve Vazektomi

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.

Hücresel dönüştürme Kaposi sarkomu Herpesvirus tarafından eğitim için bir Vitro modeli

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.

Bir hücre dışı asidik pH kültür sistemi kurulması

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.

Bir sıçan modelinde en iyi duruma getirilmiş yapısı ile Electrospun vasküler greft implantasyonu

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.

5-Aminolevulinik asit-aracılı Fotodinamik Tedavi etkisi Staphylococcus aureus biyofilm çalışmaya bir In Vitro modeli

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 etiketli CENP-A (EYFP-CENP-A) Ubiquitylation tanımlamak için Kütle Spektrometresi Analizi

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.