Yazarlar bu videoyu üretti Missouri Akademik Destek Merkezi Üniversitesi Yaratıcı Hizmetler ekibi teşekkür etmek isterdim. Bu çalışma Ulusal Sağlık Enstitüleri Çocuk Sağlığı ve İnsan Gelişimi Eunice Kennedy Shriver Ulusal Enstitüsü, hibe sayısı HD055231 tarafından desteklenmiştir

1. Fare plasentanın Diseksiyon <ol><li> Farelerde çiftleşme çiftleri ayarlayın. </li><li> İlerleyen günlerde her birinde, bir çiftleşme fiş varlığını kontrol edin. Bir fiş tespit edildiği gün 0.5 gün sonrası cinsel birleşme (DPC), 7. olarak kabul edilir. </li><li> Plasentası d 10.5 olgun plasentanın gelişimi ile plasenta mümkün başında temiz bir diseksiyon ile, gebeliğin istenen aşamada toplanabilir. Burada, 14.5 DPC göstermektedir. Verilen miktarlar ise, 10.5 DPC üzerine 40-60 plasenta ve 14.5 DPC 10-20 plasentası açıkça görülebilen bir trofoblast hücre tabakası verecektir. </li><li> Hamile fare Euthanize ve servikste kesme, rahim kaldırmak ve mesometrium boyunca. </li><li> Her konseptus (Şekil 1A) arasında kesilmiş keskin bir makas, kullanma. </li><li> Forseps, iki çift kullanarak, kesme kenarında rahim duvarına kavramak ve uzaklaşmayı, myometrium (Şekil 1B) kaldırma<html> . Bir çift tırtıklı, (kavisli veya düz) orta ince forseps artı bir çift çok ince forseps (# 5, örneğin) tavsiye edilir. </li><li> Plasenta karşısında, plasenta teşhir, forseps veya makas ile desidua açın. Tek bir forseps bir çift ile uterus dokusu kavra, ayırmak için plasenta (Şekil 1C) altında başka bir forseps kaydırın. Eğer plasenta gerekli, ayrı fetüs ve fetal membranlar. Ayrıca plasental doku kaybetmeden diseksiyonu bütün desidual doku kaldırmak mümkün değildir, ancak ek desidual doku (soluk) yavaşça forseps ile soyulması ile plasenta yüzeyinden uzaklaştırılabilir. Biz daha fazla arıtma Percoll degrade üzerinde de durulacaktır. </li><li> Her diseksiyonlar bitene kadar 50 ml konik bir tüp içinde 25 ml buz gibi soğuk tampon içinde her bir ayrılma parçalara plasenta yerleştirin. Daha sonraki aşamalarda, iri ayrışma tampon içine aktarmak için önce makas veya jilet ile plasental doku kıyma. </li></ol>trofoblast hücrelerin alışını &quot;&gt; 2. İzolasyonu <ol><li> 45-60 dakika için bir 37 ° C banyo içinde gevşek bir şekilde kapak konik boru ve bir yer. Yaklaşık her 10 dk pipetleme tarafından şiddetle ayrışma çözüm karıştırın. Toplam sindirim süresi yakından izlenmeli ve muhtemelen bazı deneme yanılma optimum hücre iyileşme elde etmek isteyecektir. Dokular 10 ml pipet geçirilecek gerekir, ancak doku görünür parçaları kalır. Overdigestion zayıf hücre hayatta kalma ve kaplama verimi ile sonuçlanır. Birkaç hücre kümeleri mevcut olduğunda Optimal hücre sağkalım ve kaplama verimliliği yerine tam ayrışma elde edildiğinde daha ortaya çıkacaktır. Tersine, underdigestion Percoll ayrışmadan sonra azalır saflığı yol açacaktır. Uygun sindirim noktasına ulaşıldığında, buz üzerinde yer tüp kollajenaz aktivitesini durdurmak için. </li><li> Sindirilmemiş malzemeleri çıkarın ve 5 dakika boyunca @ 500 xg&#39;de santrifüj bir hücre süzgecinden çözümü geçirin. </li><li> Yıkamak için kaldırmak10 ml yıkama çözeltisi içinde tekrar süspansiyon ve süpernatant hücreler. 5 dakika @ 500 xg santrifüjleyin. </li><li> Santrifüj sırasında, 9.6 ml Percoll, 13.4 ml yıkama çözeltisi ve yüksek hızlı santrifüjleme için uygun olan bir 50 ml tüp içinde 1.1 ml ortam 10x 199 karıştırılarak Percoll solüsyon hazırlanır. </li><li> 2 mL yıkama solüsyonu içinde süpernatant ve tekrar süspansiyon hücre pelletini çıkarın. </li><li> Çözelti Percoll ve 4 de 30 dakika @ 30,000 x g&#39;da santrifüj ile hücreler ekleme ° C. </li><li> Degrade bozmadan santrifüj tüpü çıkarın. Parlak bir ışık kaynağının önünde degrade, yerde tüp görselleştirmek için. Trofoblast bant (Şekil 2) bulun ve aşağıdaki kırmızı kan hücresi bant rahatsız etmemek için özen bir pipet yardımıyla aspire. Trofoblast bant hacmi 5-8 ml olacaktır. </li><li> 50 ml&#39;lik bir konik tüpe trofoblastların aktarın. 45ml toplam hacme yıkama çözeltisi ekleme. </li><li> 5 dakika @ 500 xg santrifüjleyin. Süpernatantı ve kültür medi tekrar süspansiyonum (NCTC-135). Hücreler 37 ° C&#39;de ve% 5 CO2 de ince tabaka Matrigel ya da plastik doku kültürü üzerinde kültive edilebilir. Günlük 10.5, her plasenta% 80-90 pozitif sitokeratin hangi kabaca 50.000 canlı hücreleri üretir. </li></ol> 3. Trofoblast invazyonu Assay <ol><li> 37 her Matrigel odası ve inkübe için 1-2 ml kültür ortamı (NCTC-135) ekle °, 30-90 dakika süreyle% 5 CO 2. </li><li> Orta çıkarın ve üst odacığı 2ml orta 1,5 ml x 10 5 taze izole hücreleri ekleyin. Alt membran yüzeyini kapsayacak şekilde alt kanadı 1 ml orta ekleyin. </li><li> 18-24 saat için ekler kültür hücreleri. </li><li> Bir köpük çubukla ovülden Pençe hücreleri ve Matrigel. </li><li> 2 ml PBS içinde üst ve alt bölme çalkalayın. </li><li> PBS içinde 1 ml% 4 paraformaldehit içinde 5 dakika için düzeltildi. </li><li> 400 ul DAPI (300m) ile alt kanadı doldurarak Leke ve 30 dakika inkübe edilir. </li><li> PBS içinde kısaca üç kez yıkayın. </li><li&gt; keskin bir neşter bıçağı ile ekler membranlar çıkarın. </li><li> Kaydırmak için montaj ortam maddesi (PBS,% 50 gliserol) ve bir damla 10 ul ekle. Forseps kullanarak, damla membran yatıyordu. Orta bir 10 ul damla ekleyin, sonra dikkatlice lamel. </li><li> Fotoğraf ve sayım hücreler. Kullanılan mikroskop için uygun şark membran (aşağı cep tarafını yukarı veya hücre tarafı) emin olun. Image J yazılımı (Ulusal Sağlık Enstitüleri) bu amaç için kullanılabilecek bir el sayma özelliği içerir, can Adobe Photoshop veya manuel sayaç. </li></ol> 4. Temsilcisi Sonuçlar <img src="/files/ftp_upload/3202/3202fig1.jpg" alt="Şekil 1" /> Şekil 1. Fare plasentanın Diseksiyon. (A) rahim mesometrial yüzeyi boyunca, (sarı) yumurta kanalı boyunca kesilerek çıkarılır ve serviks (alt) olarak alır. Bireysel conceptuses sonra gösterildiği gibi, makas ile kesme ile ayrılabilir. (B) rahim<html> duvar kesme rahim içinde iki yüzey forseps yerleştirerek ve fetus yakın ve uzak plasenta, anti-mesometrial tarafı boyunca yırtarak ile uzaklaştırılır. (C) Plasenta sonra forseps ile açgözlü ve plasenta ve uterus dokusu arasında bir kayma ile izole edilir. Umbilikal kord ve diğer extraembryonic membranlar daha sonra plasenta ayrılabilir. <img src="/files/ftp_upload/3202/3202fig2.jpg" alt="Şekil 2," /> Trofoblastların Şekil 2. Percoll ayrılması. Santrifüj sonra, üç büyük grup solda karikatür gösterildiği gibi görünür, ve fotoğraf sağda olacak. Üst bant, hücre tortularını ve fibroblastlar içeren ve en çok belirgin olacaktır. Kırmızı kan hücreleri içeren altına yakın Sıkı (kırmızı) bant sadece diffüz trofoblast grup altında gözükecektir. Sadece fotoğraftaki ok sol gösterilen iki gibi Hücre agrega olasılıkla, trofoblast bandında görülen olacaktır. ove_content &quot;&gt; <img src="/files/ftp_upload/3202/3202fig3.jpg" alt="Şekil 3" /> Şekil 3,. İstilacı trofoblastların (beyaz ok) zarın alt yüzeyi üzerinde görünen, Matrigel tabaka ve zar gözenekleri geçecek. Gözenekler (siyah ok başı) da görülebilir. Tüm zar yüzey ya da temsili bir numune alanına hücrelerin sayısı sayılır. Floresan DAPI nükleer boyanma bir gri görüntü hücrelerin görsel geliştirmek için ters edilmiştir. <img src="/files/ftp_upload/3202/3202fig4.jpg" alt="Şekil 4," /> Şekil 4,. Trofoblast hücreleri tespit ve izole nüfusun saflık sitokeratin 7 (merkez, macenta) ile immüno-, değerlendirilebilir. Hücreler DAPI (üst, mavi) ile zıt edilir. Birleştirilmiş görüntü, alt.

<ol>
	<li>Thordarson, G., Folger, P., Talamantes, F. <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+a+placental+cell+culture+system+for+studying+the+control+of+mouse+placental+lactogen+II+secretion.">Development of a placental cell culture system for studying the control of mouse placental lactogen II secretion.</a> Placenta. 8, 573-585 (1987).</li><li>Petroff, M. G., Phillips, T. A., Ka, H., Pace, J. L., Hunt, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+term+human+trophoblast+cells.">Isolation and culture of term human trophoblast cells.</a> Methods. Mol. Med. 121, 203-217 (2006).</li><li>Kliman, H. J., Nestler, J. E., Sermasi, E., Sanger, J. M., Strauss, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification,+characterization,+and+in+vitro+differentiation+of+cytotrophoblasts+from+human+term+placentae.">Purification, characterization, and in vitro differentiation of cytotrophoblasts from human term placentae.</a> Endocrinology. 118, 1567-1582 (1986).</li><li>Blaschitz, A., Weiss, U., Dohr, G., Desoye, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+reaction+patterns+in+first+trimester+placenta:+implications+for+trophoblast+isolation+and+purity+screening.">Antibody reaction patterns in first trimester placenta: implications for trophoblast isolation and purity screening.</a> Placenta. 21, 733-741 (2000).</li><li>Librach, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=92-kD+type+IV+collagenase+mediates+invasion+of+human+cytotrophoblasts.">92-kD type IV collagenase mediates invasion of human cytotrophoblasts.</a> J. Cell. Biol. 113, 437-449 (1991).</li><li>Schulz, L. C., Widmaier, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+leptin+on+mouse+trophoblast+cell+invasion.">The effect of leptin on mouse trophoblast cell invasion.</a> Biol. Reprod. 71, 1963-1967 (2004).</li><li>Tanaka, S., Kunath, T., Hadjantonakis, A. K., Nagy, A., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promotion+of+trophoblast+stem+cell+proliferation+by+FGF4.">Promotion of trophoblast stem cell proliferation by FGF4.</a> Science. 282, 2072-2075 (1998).</li></ol>

Biz ifşa etmek başka bir şey var.

Bu video olarak, fare plasenta trofoblast hücrelerin izolasyonu göstermektedir. Biz sonra trofoblast invazyonu için in vitro deney göstermektedir. Bu prosedür kullanıldığında, büyük ölçüde tamamen olmasa da, trofoblast hücre saf nüfus elde edilebilir. Daha fazla saflık gerekli ise primer insan trofoblast hücreleri için tarif edildiği gibi, manyetik boncuk ayırma ya da akış sitometrisi, yapılabilmektedir. Kollajenaz ayrışma adımın doğru uzunluğu her bir deney ile tespit edilmeli ve deneyimi ...

isolation of primary mouse trophoblast cells and trophoblast invasion assay

Plasenta fetusun besin, gazlar ve büyüme faktörlerinin taşıma yanı sıra, atıkların ortadan kaldırılması için sorumludur. Böylece, plasental gelişim kusurları, anne ve fetus için önemli sonuçları vardır ve embriyonik ölümcül önemli bir nedenidir. Plasenta fetal kısmının başlıca bir hücre türüdürler trofoblast olup. Birincil fare plasental trofoblast hücreleri normal ve anormal plasental geliştirme çalışmaları için yararlı bir araçtır, ve hücre çizgileri, farklı olarak, izole edilmiş ve hamilelik belirli aşamalarında trofoblast incelemek için kullanılabilir. Buna ek olarak, transgenik fareler trofoblast primer kültürlerden plasental hücreleri içinde belirli genlerin rolü incelemek için kullanılabilir. Burada sunulan protokol Thordarson ve arkadaşları tarafından açıklama esas alınmaktadır. Bir Percoll gradient izole edilmiş fare plasentası gelen, saf bir trofoblast hücre popülasyonu elde etmek için kullanıldığı, 1,. Bu, daha yaygın olarak kullanmak için benzerİnsan trofoblast hücre izolasyonu 2-3 d yöntemleri. Saflık sitokeratin 7 4 için izole hücrelerin immünohistokimyasal boyama ile değerlendirilebilir. Burada, izole hücreler daha sonra in vitro 5-6 trofoblast invazivlik değerlendirmek için bir Matrigel işgali testi kullanılarak analiz edilmiştir. Işgal hücrelerin sayımı için immünsitokimya ve lekeli tarafından analiz edilir.

Watch this Scientific Journal Video about Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay at JoVE.com

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

Bu protokol, biz bir Percoll gradyanı kullanılarak trofoblast hücrelerinin izolasyonu takiben gebelik d10.5 üzerinde fare plasenta diseksiyonu tarif. Daha sonra bir Matrigel işgali tayininde izole hücrelerin kullanımını göstermektedir.

Birincil Fare trofoblast hücreleri ve trofoblast invazyonu Assay İzolasyonu

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes.  
Thus, defects ...

isolation-primary-mouse-trophoblast-cells-trophoblast-invasion

Research

JoVE Journal

Biology

23.7K Görüntüleme.  University of Missouri. Bu protokol, biz bir Percoll gradyanı kullanılarak trofoblast hücrelerinin izolasyonu takiben gebelik d10.5 üzerinde fare plasenta diseksiyonu tarif. Daha sonra bir Matrigel işgali tayininde izole hücrelerin kullanımını göstermektedir.

Video: Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend diseases related to pregnancy. In this protocol, human primary villous cytotrophoblasts freshly isolated from placentas through a standard DNase/trypsin protocol are microporated with small interfering RNA (siRNA). This approach provided greater efficiency for siRNA transfection when compared to a lipofection-based method. Transfected cells can subsequently be analyzed by standard Western blot within a time frame of 3-4 days post-transfection. In addition, using cultured primary villous cytotrophoblasts, Electrophoretic Mobility Shift Assay (EMSA) analysis was optimized and performed on extracts from days 1 to 4. The use of these cultured primary cells and the protocol described allow for an evaluation of the implication of specific genes and transcription factors in the process of villous cytotrophoblast differentiation into a syncytiotrophoblast-like cell layer. However, the limited time span allowable in culture precludes the use of methods requiring more time, such as generation of a stable cell population. Therefore testing of this cell population requires highly optimized gene transfer protocols.

This protocol describes a method for efficiently transfecting siRNA in freshly isolated human villous cytotrophoblasts using microporation and identifying DNA-protein complexes in these cells. Transfected cells can be monitored by Western blot and EMSA analyses during the 4-day culture time.

Taze izole edilmiş insan Villöz sitotrofoblastlar ilgili siRNA transfeksiyonu ve EMSA analizi

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend ...

Genetik

Human Primary Trophoblast Cell Culture Model to Study the Protective Effects of Melatonin Against Hypoxia/reoxygenation-induced Disruption

This protocol describes how villous cytotrophoblast cells are isolated from placentas at term by successive enzymatic digestions, followed by density centrifugation, media gradient isolation and immunomagnetic purification. As observed in vivo, mononucleated villous cytotrophoblast cells in primary culture differentiate into multinucleated syncytiotrophoblast cells after 72 hr. Compared to normoxia (8% O2), villous cytotrophoblast cells that undergo hypoxia/reoxygenation (0.5% / 8% O2) undergo increased oxidative stress and intrinsic apoptosis, similar to that observed in vivo in pregnancy complications such as preeclampsia, preterm birth, and intrauterine growth restriction. In this context, primary villous trophoblasts cultured under hypoxia/reoxygenation conditions represent a unique experimental system to better understand the mechanisms and signalling pathways that are altered in human placenta and facilitate the search for effective drugs that protect against certain pregnancy disorders. Human villous trophoblasts produce melatonin and express its synthesizing enzymes and receptors. Melatonin has been suggested as a treatment for preeclampsia and intrauterine growth restriction because of its protective antioxidant effects. In the primary villous cytotrophoblast cell model described in this paper, melatonin has no effect on trophoblast cells in normoxic state but restores the redox balance of syncytiotrophoblast cells disrupted by hypoxia/reoxygenation. Thus, human villous trophoblast cells in primary culture are an excellent approach to study the mechanisms behind the protective effects of melatonin on placental function during hypoxia/reoxygenation.

This manuscript presents a unique in vitro model of immunopurified human villous cytotrophoblast cells cultured under hypoxia/reoxygenation. This model is suitable to study the protective effects of promising treatments, such as melatonin, on pregnancy complications associated with increased oxidative stress and altered placental function.

İnsan İlköğretim Trofoblast Melatonin Karşı Hipoksi Koruyucu Etkilerinin Eğitim için Hücre Kültürü Modeli / reoksijenasyon kaynaklı bozulması

This protocol describes how villous cytotrophoblast cells are isolated from placentas at term by successive enzymatic digestions, followed by density ...

Gelişim Biyolojisi

Protocol for the Direct Conversion of Murine Embryonic Fibroblasts into Trophoblast Stem Cells

Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining the ability to self-renew and to differentiate into all subtypes of the trophoblast lineage, equivalent to the in vivo stem cell population giving rise to the fetal portion of the placenta. Therefore, TSCs offer a unique model to study placental development and embryonic versus extra-embryonic cell fate decision in vitro. From the blastocyst stage onwards, a distinct epigenetic barrier consisting of DNA methylation and histone modifications tightly separates both lineages. Here, we describe a protocol to fully overcome this lineage barrier by transient over-expression of trophoblast key regulators Tfap2c, Gata3, Eomes and Ets2 in murine embryonic fibroblasts. The induced trophoblast stem cells are able to self-renew and are almost identical to blastocyst derived trophoblast stem cells in terms of morphology, marker gene expression and methylation pattern. Functional in vitro and in vivo assays confirm that these cells are able to differentiate along the trophoblast lineage generating polyploid trophoblast giant cells and chimerizing the placenta when injected into blastocysts. The induction of trophoblast stem cells from somatic tissue opens new avenues to study genetic and epigenetic characteristics of this extra-embryonic lineage and offers the possibility to generate trophoblast stem cell lines without destroying the respective embryo.

Here we present a protocol for the direct conversion of murine embryonic fibroblasts into fully functional and stable trophoblast stem cells by ten day over-expression of Tfap2c, Gata3, Eomes and Ets2.

Trofoblast Kök Hücre içine Fare Embriyonik Fibroblastlar Doğrudan Dönüşüm Protokolü

Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

Insan pluripotent İn Vitro Farklılaşma Trofoblastik Hücreleri içine Kök Hücreler

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

The Use of Mouse Mammary Tumor Cells in an In Vitro Invasion Assay as a Measure of Oncogenic Cell Behavior

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a porous membrane in a process analogous to the initial steps of cancer cell metastasis. The tested cells can be altered for the gene expression or treated with inhibitors to test for changes in the invasion potential. This experiment tests the aggressive phenotype of the mouse mammary tumor cells to discover and characterize the potential oncogenes that promote cell invasion. This technique, however, can be versatile and adapted to many different applications. The experiment itself can be done in one day and the results are acquired by light microscopy in less than a day. The results include counts of the number of invading cells for comparison and analysis. The in vitro invasion assay is a rapid, inexpensive, and clear-cut method for determining cell behavior in a culture that can be used as an initial assessment before more involved in vivo assays.

The in vitro cell invasion assay is used to measure the potential of cancer metastasis by quantifying the cellular potential for invasion and migration using cell culture inserts containing protein matrix. Cells are challenged to migrate through the protein matrix and a porous membrane, towards a chemoattractant, and then quantified by light microscopy.

Onkojenik hücre davranışının bir ölçüsü olarak bir In vitro Invazif tahlil içinde Mouse mammary tümör hücrelerinin kullanımı

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a ...

In Vitro Assays to Evaluate the Migration, Invasion, and Proliferation of Immortalized Human First-trimester Trophoblast Cell Lines

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration and invasion is demonstrated by pregnancy disorders such as pre-eclampsia and intrauterine growth restriction, which are associated with inadequate trophoblast invasion of the maternal vasculature. Unfortunately, our understanding of the mechanisms by which the placenta develops from migrating trophoblasts is limited. In vitro analysis of cell migration via the scratch assay is a useful tool in identifying factors that regulate trophoblast migratory capacity. However, this assay alone does not define the cellular changes that can result in altered cell migration. This protocol describes three different in vitro assays that are used collectively to evaluate trophoblast cell movement: the scratch assay, the invasion assay, and the proliferation assay. The protocols described here may also be modified for use in other cell lines to quantify cell movement in response to stimuli. These methods allow investigators to identify individual factors that contribute to the cell movement and provide a thorough examination of potential mechanisms underlying apparent changes in cell migration.

Here, we present a highly accessible protocol for evaluating the cell movement in human trophoblast cells using three in vitro assays: the scratch assay, the transwell invasion assay, and the cell proliferation assay.

Vitro deneyleri içinde geçiş, işgal ve ölümsüzleştirdi insan birinci üç aylık dönem Trofoblast hücre hatları yaygın değerlendirmek için

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration ...

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the trophectoderm mediates implantation and generates the fetal part of the placenta. As a result, the development of this lineage is essential for embryo survival. Derivation of trophoblast stem (TS) cells from mouse blastocysts was first described by Tanaka et al. 1998. The ability of TS cells to preserve the trophoblast specific property and their expression of stage- and cell type-specific markers after proper stimulation provides a valuable model system to investigate trophoblast lineage development whereby recapitulating early placentation events. Furthermore, trophoblast cells are one of the few somatic cell types undergoing natural genome amplification. Although the molecular pathways underlying trophoblast polyploidization have begun to unravel, the physiological role and advantage of trophoblast genome amplification remains largely elusive. The development of diploid stem cells into polyploid trophoblast cells in culture makes this ex vivo system an excellent tool for elucidating the regulatory mechanism of genome replication and instability in health and disease. Here we describe a protocol based on previous reports with modification published in Chiu et al. 2008.

In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.

Blastosist Fare trofoblast Kök Hücre türetilmesi

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...

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Analyzing Angiogenesis: In Vitro Assay for Tube Formation on BM Matrix

Trophoblast Cell Recovery from Angiogenesis-Tube Formation Assay for Differentiation Marker Expression Analysis

During placenta development, extravillous trophoblast (EVT) cells invade the maternal decidua to remodel the uterine spiral arteries by a process of mesenchymal to endothelial-like transition. Traditionally, this process is evaluated by an in vitro tube-formation assay, where the cells organize themselves into tube-like structures when seeded over a polymerized basement membrane preparation. Although several structural features can be measured in photomicrographs of the structures, to assess the real commitment of EVT to the endothelial-type phenotype, biochemical analysis of cell extracts is required. Scraping the cells from the culture dish to obtain RNA and/or protein extracts is not an alternative since the tube-like structures are severely contaminated by the bulk of proteins from the polymerized basement membrane. Thus, a strategy to separate the cells from the basement membrane proteins prior to the preparation of cell extracts is needed. Here, a simple, fast, and cost-effective method to recover the tube-like structures from the in vitro tube-formation assay and the subsequent analysis by biochemical techniques is presented. Tube-like structures formed by HTR8/SVneo cells, an EVT cell line, were liberated from the polymerized basement membrane by a short incubation with PBS supplemented with ethylenediaminetetraacetic acid (EDTA). After serial washes, a ready-to-use pellet of purified tube-like structures can be obtained. This pellet can be subsequently processed to obtain RNA and protein extracts. qPCR analysis evidenced the induced expression of VE-cadherin and alphav-integrin, two endothelial cell markers, in EVT-derived tube-like structures compared to control cells, which was consistent with the induction of the endothelial cell marker, CD31, evaluated by immunofluorescence. Western blot analysis of the tube-like structures' protein extracts revealed the overexpression of RECK in transfected HTR8/SVneo cells. Thus, this simple method allows to obtain cell extracts from the in vitro tube-formation assay for the subsequent analysis of RNAs and protein expression.

The present protocol allows the recovery of tube-like structures formed in the classical in vitro angiogenesis-tube formation assay where cells, such as trophoblast cells, organize in capillary-like structures over a layer of extracellular basement membrane matrix. The recovered structures are used for RNA and protein isolation for various biochemical analyses.

During placenta development, extravillous trophoblast (EVT) cells invade the maternal decidua to remodel the uterine spiral arteries by a process of ...

A Primary Human Trophoblast Model to Study the Effect of Inflammation Associated with Maternal Obesity on Regulation of Autophagy in the Placenta

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be attributed to, in part, the dysregulation of autophagy. Aberrant changes in the expression of autophagy regulators in the placentas from obese pregnancies may be regulated by inflammatory processes associated with both obesity and pregnancy. Described here is a protocol for sampling of villous tissue and isolation of villous cytotrophoblasts from the term human placenta for primary cell culture. This is followed by a method for simulating the inflammatory milieu in the obese intrauterine environment by treating primary trophoblasts from lean pregnancies with tumor necrosis factor alpha (TNF&#945;), a proinflammatory cytokine that is elevated in obesity and in pregnancy. Through the implementation of the protocol described here, it is found that exposure to exogenous TNF&#945; regulates the expression of Rubicon, a negative regulator of autophagy, in trophoblasts from lean pregnancies with female fetuses. While a variety of biological factors in the obese intrauterine environment maintain the potential to modulate critical pathways in trophoblasts, this ex vivo system is especially useful for determining if expression patterns observed in vivo in human placentas with maternal obesity are a direct result of TNF&#945; signaling. Ultimately, this approach affords the opportunity to parse out the regulatory and molecular implications of inflammation associated with maternal obesity on autophagy and other critical cellular pathways in trophoblasts that have the potential to impact placental function.

Presented here is a protocol for sampling of human placental villous tissue followed by isolation of cytotrophoblasts for primary cell culture. Treatment of trophoblasts with TNF&#945; recapitulates inflammation in the obese intrauterine environment and facilitates the discovery of molecular targets regulated by inflammation in placentas with maternal obesity.

Plasenta Autophagy düzenlenmesi üzerinde anne obezite ile ilişkili iltihap etkisini incelemek için bir birincil insan Trofoblast modeli

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be ...

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Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized stromal cells and immune cells, are responsible for the secretion of hormonal and inflammatory factors which are critical for successful blastocyst implantation, placental development and play a role in the initiation of labor at term and preterm. Many pregnancy complications can arise from perturbations of a fine balance of different cell populations comprising decidua. Alterations in the proportion of specific decidual cell types may disrupt these crucial processes and increase the risk of developing serious complications of pregnancy, such as embryo implantation failure, intrauterine growth restriction, preeclampsia and preterm labor. The protocol outlined here demonstrates a cost and time effective method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae. By combining enzymatic digestion and gentle mechanical disruption of the decidual tissue, a high yield of decidual cells was obtained with virtually no chorion contamination. Importantly, isolated decidual cells were characterized (stromal cells (55-60%), leukocytes (35%), epithelial (1%) or trophoblast (0.01%) cells) and maintained high viability (80%) which was confirmed by multicolor imaging flow cytometry assay. This protocol is specific to the decidua parietalis and can be adapted to first and second trimester placentae. Once isolated, decidual cells can be used for a multitude of experimental applications aiming to understand the role of different decidual cell sub-populations in pregnancy complications.

This protocol demonstrates a method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae which can be used for a variety of applications (i.e. immunocytochemistry, flow cytometry, etc.) aiming to study the role of different cell populations in pregnancy complications.

Dönem Placentae Fetal membranlar birincil insan Decidual hücre izolasyon

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...