作者要感谢捐助者、RCWIH 生物库和西奈医院/--减压振动妇产科为本研究使用的人体标本。我们要感谢碱液实验室的成员, 特别是卡罗琳·扣篮博士对她的方法开发的帮助。这项工作得到了宝来欢迎基金 (赠款 #1013759) 的支持。

胎盘是从健康的术语中收集的, 而不是在接受选择性剖宫产的分娩妇女身上。人体样品的收集、处理和一次性使用遵循西奈医院伦理委员会的指导方针。每个病人都能得到书面同意。这项研究由西奈山医院的研究伦理学委员会批准。
1. 筹备工作
注: 所有步骤必须在通风罩下进行, 所有手术设备必须在放置在通风罩前通过高压釜消毒。所有其他材料 (瓶, 50 毫升管,等) 必须用70% 乙醇溶液灭菌。在与 biohazardous 废物 (实验室大衣, 手套, 长头发绑回,等) 工作时, 始终佩戴个人防护设备。
<ol><li>
酶消化溶液 (最终溶液: 200 毫升)
	<ol>
<li>吸管180毫升的 HBSS-/-成500毫升烧杯, 加入无菌搅拌磁铁, 并放置在搅拌板室温。</li>
		<li>称量并添加以下的 HBSS/-解决方案缓慢和依次:20 毫升的血清, 400 毫克胶原酶 2 ([最终] = 2 毫克/毫升), 20 毫克大豆胰蛋白酶抑制剂 ([最终] = 0.1mg/毫升), 30 毫克 DNase 1 ([最终] = 0.15 毫克/毫升), 200 毫克 BSA ([最终] = 1 毫克/毫升)</li>
		<li>将搅拌速度设置为中速, 并允许混合物搅拌10-20 分钟 (在搅拌时用锡纸或石蜡膜覆盖)。</li>
		<li>将混合溶液倒入一个250毫升的玻璃瓶, 使用塑料漏斗和放置在通风罩。</li>
		<li>通过一个塑料顶部过滤单元 (0.22 微米膜过滤器, 500 毫升), 整除20毫升到50毫升管 (10 管共计), 并存储在-20 °c, 直到使用的解决方案。</li>
	</ol>
</li>
	<li>
RPMI 1640 介质 (2% 洗涤液和10% 完全生长培养基)
	<ol>
<li>结合 RPMI 1640 和在一个玻璃瓶中的烟雾罩。</li>
		<li>对于2% 的血清溶液, 结合490毫升的 RPMI 1640 和10毫升的血清。</li>
		<li>对于10% 的血清溶液, 结合450毫升 RPMI 1640 和50毫升的血清。</li>
		<li>吸管 500 ul normocin 进入500毫升玻璃瓶从前一步包含 RPMI 媒体和血清 (50 毫克/毫升库存, 0.05 毫克/毫升工作浓度)。</li>
		<li>通过0.22 微米膜过滤器和存储在500毫升玻璃瓶在4°c, 直到使用。</li>
	</ol>
</li>
	<li>30分钟开始实验前, 在37°c 的珠浴中放置20毫升的冷冻酶消化液整除, 在10% 摄氏度的珠浴中放置2% 的血清和1640的 RPMI 37 介质, 打开摇摆水浴, 设置为37°c。, 2 克, 并设置温度控制离心机到4摄氏度。</li>
</ol>2. 从足月胎盘膜中收集蜕组织
<ol><li>将瓶子与 HBSS +/+, HBSS 和五50毫升管在一个机架下的通风罩。在一管中吸管25毫升的 HBSS。</li>
	<li>
使用戴手套的手, 从使用的容器中取出胎盘, 从手术室剧院运送。将胎盘放在尿布垫上 (孕产妇侧), 用剪刀和镊子将胎儿膜张开。
	<ol>
<li>在通风罩上放置重叠的尿布垫。</li>
		<li>使用一个单独的尿布翻转胎盘, 使产妇一侧脸上。</li>
		<li>找出膜破裂的点, 使切口允许膜在通风橱表面展开和平躺。 注: 一旦膜从胎盘中拉回, 将会在绒毛膜层上, 将羊膜层放在后面。</li>
	</ol>
</li>
	<li>使用塑料细胞刮刀, 仔细刮蜕组织关闭绒毛膜和地方在50毫升管包含25毫升 HBSS ++。</li>
	<li>用中等压力刮膜。不要施加太多的压力, 因为它可能导致绒毛膜污染。收集小血块以及, 因为这些含有一些蜕细胞。 注意: 在蜕收集后, 胎盘必须包装在一个生物危害塑料箱和冻结在-20 摄氏度冷藏之前, 适当处置 (根据您的机构规则)。血淋淋的尿布垫必须包裹在密封紧的塑料袋中, 并处置在一个生化安全箱。</li>
</ol>3. 蜕组织的洗涤和酶消化
<ol><li>用手轻轻摇动50毫升管, 通过250微米金属筛 (250 微米, 大小60目) 在无菌标本 (尿) 容器上, 冲洗收集的组织。</li>
	<li>重复洗两次与 HBSS +/+ 两次与 HBSS-/在新鲜管每洗。(HBSS 的最终洗涤--允许去除 HBSS 中存在的钙和镁, 否则会干扰下面的酶消化过程)。 注意。在洗涤步骤, 绒毛膜组织污染将明显, 因为蜕组织是淡粉红色的颜色和无定形, 而绒毛膜是白色, 致密, 和粘性。因此, 绒毛膜污染可以很容易地去除与镊子。</li>
	<li>如果蜕组织是厚的, 将其转移到一个不育的10厘米直径培养皿, 并进行的组织与两个对立的手术刀在盘子里的肉。</li>
	<li>
将洗涤后的组织放置在50毫升的无菌管中, 含有100毫克的酶消化溶液 (见制剂)。
	<ol>
<li>作为参考点, 确保蜕组织的水平达到50毫升管的5-10 毫升标记。</li>
		<li>使用大约20毫升的酶消化溶液 (在步骤1.1 中制备), 以消化从一个完整的足月胎儿膜中收集的全膜。</li>
	</ol>
</li>
	<li>用石蜡膜封住管帽 (密封管, 并在管帽和顶部周围涂上石蜡膜), 并在37摄氏度的水浴中孵化蜕组织20分钟 (145 rpm, 2 克)。</li>
	<li>孵化后, 取出石蜡膜, 用70% 乙醇将含有消化组织的管表面消毒, 并将其置于油烟罩下。用手简单地摇动管子。</li>
	<li>收集细胞悬浮通过金属筛 (250 微米, 大小60目) 成一个新的无菌标本容器。稀释与等量 (20 毫升) 的 RPMI + 10% 的血清含有 0.1% normocin, 以停止酶反应。直接进行离心步骤4.1。</li>
	<li>如有必要, 将余下未消化的组织放回新的50毫升管中, 20 毫升新鲜酶消化溶液和重复消化 (20 分钟, 37 °c, 摇动水浴)。</li>
	<li>如果第二次消化是必要的, 放置第一管与细胞悬浮在冰 (覆盖与石蜡膜保持无菌)。重复步骤 3.5-3.6, 并结合两个细胞悬浮。</li>
</ol>4. 产生单个细胞悬浮液
<ol><li>离心机细胞悬浮液 (420 克, 4 °c, 11 分钟)。</li>
	<li>移除上清和并用重悬40毫升的 RPMI + 2% 的血清中含有 0.1% normocin ("洗涤缓冲") 的细胞。 注: 细胞颗粒会因红细胞污染而松动和凝胶, 并慎吸上清液。可能需要使用手动吸管移除上部相。</li>
	<li>重复离心在420克在4°c 11 分钟。</li>
	<li>小心移除上清液 (如注步骤4.2 中所述), 并并用重悬5毫升洗涤缓冲器中的细胞颗粒, 并在同一管中添加35毫升的红细胞溶解缓冲液。 注: 如果颗粒大或非常血腥, 可将10毫升的洗涤缓冲液分成两个管, 然后将35毫升的红细胞溶解缓冲液分给各管, 以将其分解为红细胞裂解步骤。</li>
	<li>在冰上孵育20分钟, 短暂的旋涡管在孵化开始和结束时溶解红细胞。</li>
	<li>离心机在420克11分钟4摄氏度。</li>
	<li>小心, 取出上清和并用重悬40毫升的洗涤缓冲颗粒。</li>
	<li>通过70微米的尼龙过滤器来去除细胞团簇。</li>
	<li>离心机在420克, 11 分钟4摄氏度。</li>
	<li>取出上清液, 并用重悬10毫升的全培养基 (RPMI 10% + 0.1% normocin) 的细胞颗粒。</li>
	<li>
使用台盼蓝染料排除 hemocytometer 程序计数单元格, 如下所述:
	<ol>
<li>在培养罩下轻轻吸管细胞悬浮向上和向下 3x, 以彻底混合前与台盼蓝结合。在1.5 毫升管准备细胞悬浮, 稀释 1:2 (结合 20 ul 的台盼蓝溶液和 20 ul 的蜕细胞悬浮)。仔细混合的台盼蓝细胞解决方案通过吹打上下两次 (这个解决方案是不育)。</li>
		<li>将 hemocytometer 放在显微镜的舞台上, 玻璃盖玻片在上面。 注: Hemocytometer 是一个显微镜幻灯片与网格在它给九大正方形除以三重线。每个大正方形的面积为1毫米2, 腔内流体的深度为0.1 毫米。因此, 可以填充每个大正方形的流体体积是 1 mm*1 mm * 0.1 毫米 = 0.1 毫米3= 10-4 mL。</li>
		<li>慢慢地将台盼蓝细胞混合物的 10 ul 添加到 hemocytometer 的凹槽中, 允许毛细管作用将细胞混合物分散在整个滑梯上 (在混合物填充好之前停止)。</li>
		<li>查看显微镜下的细胞 (在10X 放大倍数) 和计数所有的白色/绿色的细胞, 排除台盼蓝 (这些是可行的细胞) 在四大的外广场的 hemocytometer。当计算接触线的单元格时, 只计算那些接触到右边和上面的线, 而不是那些接触到左和下线的细胞。不要数深蓝色细胞;蓝颜色表明, 当台盼蓝染料很容易穿透细胞膜进入细胞质时, 细胞就会死亡。</li>
		<li>计算1毫升细胞悬浮液中存活细胞的数量 (X): <img alt="Equation" src="/files/ftp_upload/57443/57443eq1v2.jpg"> 2 =稀释因子 1万 = 转换因子 (1 毫升 = 1 cm3 = 1万 * 0.1 mm3)</li>
	</ol>
</li>
	<li>稀释蜕细胞到一个理想的最终浓度在 RPMI + 10% 血清 (生长培养基)。 注: 为组织培养电镀, 吸管 2 * 106细胞/井入塑料6井板材, 10 * 106细胞入 10 cm 塑料板材或7.5万个细胞到玻璃盖玻片。</li>
</ol>

<ol>
	<li>Brosens, N., Hayashi, N., White, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progesterone+receptor+regulates+decidual+prolactin+expression+in+differentiating+human+endometrial+stromal+cells.">Progesterone receptor regulates decidual prolactin expression in differentiating human endometrial stromal cells.</a> Endocrinology. 140, 4809-4820 (1999).</li><li>Bell, S. C., D'Arcangues, C., Frase, I. S., Newton, J. R., Odlind, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Decidualization and relevance to menstruation. , 187-212 (1990).</li><li>Kariya, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-1+inhibits+in+vitro+decidualization+of+human+endometrial+stromal+cells.">Interleukin-1 inhibits in vitro decidualization of human endometrial stromal cells.</a> Journal of Clinical Endocrinology and Metabolism. 73, 1170-1174 (1991).</li><li>Dimitriadis, E., Robb, L., 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=Interleukin+11+advances+progesterone-induced+decidualization+of+human+endometrial+stromal+cells.">Interleukin 11 advances progesterone-induced decidualization of human endometrial stromal cells.</a> Molecular and Human Reproduction. 8, 636-643 (2002).</li><li>Wu, W. -. X., Brooks, J., Glasier, A. F., McNeilly, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+decidualization+and+prolactin+mRNA+and+production+at+different+stages+of+human+pregnancy.">The relationship between decidualization and prolactin mRNA and production at different stages of human pregnancy.</a> Society for Endocrinology. 14, 255-261 (1995).</li><li>Bell, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+secretion+of+protein+by+the+endometrium+and+decidua.">Synthesis and secretion of protein by the endometrium and decidua.</a> Implantation: Biology and Clinical Aspects. , 95-118 (1988).</li><li>Croy, B. A., Chantakru, S., Esadeg, S., Ashkar, A. A., Wei, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decidual+natural+killer+cells:+key+regulators+of+placental+development.">Decidual natural killer cells: key regulators of placental development.</a> Journal of Reproductive Immunology. 57, 151-168 (2002).</li><li>Smarason, A. K., Gunnarsson, A., Alfredsson, J. H., Valdimarsson, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocytosis+and+monocytic+infiltration+of+decidua+in+early+pregnancy.">Monocytosis and monocytic infiltration of decidua in early pregnancy.</a> Journal of Clinical and Laboratory Immunology. 21, 1-5 (1986).</li><li>Daiter, E., Pampfer, S., Yeung, Y. G., Barad, D., Stanley, E. R., Pollard, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+colony-+stimulating+factor-1+in+the+human+uterus+and+placenta.">Expression of colony- stimulating factor-1 in the human uterus and placenta.</a> Journal of Clinical Endocrinology and Metabolism. 74, 850-858 (1992).</li><li>Casey, M. L., Cox, S. M., Beutler, B., Milewich, L., MacDonald, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cachectin/tumor+necrosis+factor-alpha+formation+in+human+decidua.+Potential+role+of+cytokines+in+infection-induced+preterm+labor.">Cachectin/tumor necrosis factor-alpha formation in human decidua. Potential role of cytokines in infection-induced preterm labor.</a> Journal of Clinical Investigation. 83, 430-436 (1989).</li><li>Lala, P. K., Kennedy, T. G., Parhar, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+lymphocyte+alloreactivity+by+early+gestational+human+decidua.+II.+Characterization+of+the+suppressor+mechanisms.">Suppression of lymphocyte alloreactivity by early gestational human decidua. II. Characterization of the suppressor mechanisms.</a> Cellular Immunology. 127, 368-381 (1988).</li><li>Shynlova, O., Nedd-Roderique, T., Li, Y., Dorogin, A., Nguyen, T., Lye, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infiltration+of+myeloid+cells+into+decidua+is+a+critical+early+event+in+the+labour+cascade+and+post-partum+uterine+remodelling.">Infiltration of myeloid cells into decidua is a critical early event in the labour cascade and post-partum uterine remodelling.</a> Journal of Cellular and Molecular Medicine. 17, 311-324 (2013).</li><li>Osman, I., Young, A., Ledingham, M. A., Thomson, A. J., Jordan, F., Greer, I. A., Norman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+density+and+pro-inflammatory+cytokine+expression+in+human+fetal+membranes,+decidua,+cervix+and+myometrium+before+and+during+labour+at+term.">Leukocyte density and pro-inflammatory cytokine expression in human fetal membranes, decidua, cervix and myometrium before and during labour at term.</a> Molecular Human Reproduction. 9, 41-45 (2003).</li><li>Farine, T., Lye, S. J., Shynlova, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+maternal+leukocytes+are+activated+in+response+to+cytokines+secreted+by+uterine+tissues+of+pregnant+women.">Peripheral maternal leukocytes are activated in response to cytokines secreted by uterine tissues of pregnant women.</a> Journal of Cellular and Molecular Immunology. 14, 635-638 (2017).</li><li>Hamilton, S., 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=Macrophages+infiltrate+the+human+and+rat+decidua+during+term+and+preterm+labor:+evidence+that+decidual+inflammation+precedes+labor.">Macrophages infiltrate the human and rat decidua during term and preterm labor: evidence that decidual inflammation precedes labor.</a> Biology of Reproduction. 86, 39 (2011).</li><li>Casey, M. L., Cox, S. M., Word, A., Macdonald, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokines+and+infection-induced+preterm+labour.">Cytokines and infection-induced preterm labour.</a> Reprodution Fertility and Development. 2, 499-510 (1990).</li><li>Yellon, S. M., Mackler, A. M., Kirby, M. A. <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+leukocyte+traffic+and+activation+in+parturition.">The role of leukocyte traffic and activation in parturition.</a> Journal of the Society for Gynecologic Investigation. 10, 323-338 (2003).</li><li>Gomez-Lopez, N., StLouis, D., Lehr, M. S., Sanchez-Rodriguez, E. N., Arenas-Hernandez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+cells+in+term+and+preterm+labor.">Immune cells in term and preterm labor.</a> Cellular &amp; Molecular Immunology. 11, 571-581 (2014).</li><li>Xu, Y., Plazyo, O., Romero, R., Hassan, S. S., Gomez-Lopez, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+leukocytes+from+the+human+maternal-fetal+interface.">Isolation of leukocytes from the human maternal-fetal interface.</a> Journal of Visualized Experiments. 99, (2015).</li><li>Trundley, T., Gardner, L., Northfield, J., Moffett, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+isolation+of+cells+from+the+human+fetal-maternal+interface.">Methods for isolation of cells from the human fetal-maternal interface.</a> Methods in Molecular Medicine. 122, 109-122 (2006).</li><li>Jividen, K., Movassagh, M. J., Jazaeri, A., Li, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+methods+for+establishing+primary+human+endometrial+stromal+cells+from+hysterectomy+specimens.">Two methods for establishing primary human endometrial stromal cells from hysterectomy specimens.</a> Journal of Visualized experiments. 87, (2014).</li><li>Pelekanos, R. A., Sardesai, V. S., Futrega, K., Lott, W. B., Kuhn, M., Doran, M. R. <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+expansion+of+mesenchymal+stem/stromal+cells+derived+from+human+placenta+tissue.">Isolation and expansion of mesenchymal stem/stromal cells derived from human placenta tissue.</a> Journal of Visualized experiments. 112, (2016).</li><li>De Clercq, K., Hennes, A., Vrien, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mouse+endometrial+epithelial+and+stromal+cells+for+in+vitro+decidualization.">Isolation of mouse endometrial epithelial and stromal cells for in vitro decidualization.</a> Journal of Visualized Experiments. 121, (2017).</li><li>Zhang, J., Shynlova, O., Sabras, S., Bang, A., Briollais, L., Lye, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunophenotyping+and+activation+status+of+maternal+and+peripheral+blood+leukocytes+during+pregnancy+and+labour,+both+term+and+preterm.">Immunophenotyping and activation status of maternal and peripheral blood leukocytes during pregnancy and labour, both term and preterm.</a> Journal of Cellular and Molecular Medicine. 10, 2386-2402 (2017).</li></ol>

作者没有什么可透露的

此处描述的协议说明了一种成本和时间有效的方法, 用于隔离从整个人类足月胎盘的胎儿细胞膜中收集的主要蜕细胞, 这是非常容易接近和直接的。该协议的成功取决于两个关键因素, (1) 蜕刮的效率从绒毛膜层的胎儿膜和 (2) 照顾, 蜕细胞处理整个议定书。重要的是, 绒毛膜组织污染的控制, 确保没有意外包括在蜕刮, 并确定和清除任何污染在洗涤步骤 (蜕膜和绒毛膜形态的差异允许容易去除的绒毛膜, 可能是无意中收集的蜕膜)。蜕细胞是脆弱的因此快速和频繁吹打可能导致减少的生存能力。我们建议, 然后, 吸管喷射器的速度设置应该保持在缓慢和最小的吹打应使用时, 混合细胞。
本议定书防止滋养层污染的方法之一是仅从胎盘的胎儿细胞膜上收集蜕膜 (包括胎膜颞骨), 不包括通过活检进行的基底的收集。从胎盘的母体侧面。因此, 虽然我们目前的协议提高了我们分离的蜕细胞的纯度, 但它确实排除了蜕膜 (即蜕膜基底) 的一部分, 这可能会成为针对蜕膜的这一部分的研究的限制, 或者当两者都部分的蜕膜是必需的。这种排除包括这项研究的最大限制。此外, 隔离蜕细胞从人的病人允许更具生物学意义的洞察到蜕和妊娠研究, 较少的操作和更少的时间段可以研究与啮齿动物为基础的研究, 由于伦理原因。
在整个议定书中, 有一些步骤可能会造成困难, 如果不能适当减轻, 可能会影响细胞的产量和生存能力。为了环游这一点, 可能需要进行修改。最关键的是要确保蜕刮出的胎儿膜是足够的 (步骤 2.4);由于人类的变异, 每个胎盘在蜕组织的数量、组织的一致性和组织的干燥程度方面可能有所不同。重要的是调节刮擦, 以确保只有蜕膜被删除。如果蜕膜和薄膜干燥, PBS/-可以倒在膜上, 以便更容易刮。第二步是蜕细胞悬浮液的红细胞裂解 (步骤 3.4)。如前所述, 如果细胞颗粒很大, 含有大量的血液, 则可以将颗粒分成两个管, 以提高株溶藻的效率。如果这一步之后还有红细胞, 可能需要将小球分成更多的管子, 以达到最大的效率。
虽然不同的蜕隔离协议, 从足月胎盘和分离的子宫内膜细胞从非怀孕妇女的子宫切除术已经发布18, 19, 这些方法不解决各种限制, 如低细胞活力和相邻的组织污染。在设计本协议时, 我们考虑了这些限制, 从而导致细胞产量增加, 生存能力提高, 并减少了周围组织的污染风险。以前, 自动 dissociations 已用于机械地分解蜕组织19,20。这种方法是相对苛刻的, 因为它包括一个强有力的机械分离, 在蜕组织收集在一个管, 插入到机器中的功能类似的搅拌机通过快速旋转剪分解组织, 导致细胞活力降低。我们这里描述的新方法结合了短 (20 分钟) 酶消化和柔和的摇摆水浴运动, 而不是机械力应用于组织。酶消化液用于提高细胞活力和产量, 含 (1) 胶原酶消化蜕组织细胞外基质;(2) 大豆胰蛋白酶抑制剂, 防止由于胶原酶的残留 trypsinolytic 活性而导致的非特异性消化;(3) DNase 1 去除游离漂浮 DNA (4) 胎牛血清 (血清) 和牛血清白蛋白 (BSA) 蛋白, 以保护细胞不受过度消化。前一项研究的第二个主要局限在于, 通过切割胎盘母体一侧的上层来收集蜕膜基底, 这可能会导致滋养细胞的污染。我们的新协议分离的蜕膜颞骨通过轻轻地刮脱落的胎膜 (绒毛膜), 并允许在洗涤步骤中容易去除绒毛膜污染。
因此, 我们的方法允许在短时间内从胎盘膜中分离出一个简单的蜕细胞 (整个过程需要大约3小时), 从而导致高细胞产量和良好的细胞活力。一旦蜕细胞被隔离, 它们就可以用于大量的实验;例如, 人类主要的蜕细胞可以在盖玻片上播种, 以进行细胞化学分析, 它们可以生长在不同的治疗和操作的文化中, 以及直接染色的多色流式细胞术分析, 以评估人蜕膜的组成与妊娠并发症, 试图了解不同细胞亚群的作用。虽然这里提出的方法是用来隔离足月胎盘的细胞 (人类妊娠的晚期), 这个协议也可以很容易地适应第一和第二胎胎盘允许妊娠期的灵活性那个人的研究正在解决。
以前发布的协议使用自动 dissociations 分开蜕组织或酵素消化单独21,22。通过结合一个温和的机械力 (摇摆水浴在37°c) 以及酶消化鸡尾酒设计, 以优化消化和细胞的生存能力, 上面概述的协议, 创造了理想的平衡细胞产量和生存能力。此外, 以前的协议没有提到从它们的隔离获得的总细胞产量, 而且只有目前的细胞生存率为19。在这里, 我们显示了通过细胞计数确定的蜕细胞的高产量 (从10-20 每胎盘的范围不等)。此外, 目前的许多出版物表明, 从小鼠中分离出初级蜕/子宫内膜细胞;我们的协议允许对人体组织进行研究, 使它成为实验23的更具生物学意义的细胞来源。这项协议也可以很容易地适应第一和第二胎胎盘允许的妊娠阶段的灵活性, 你的研究正在处理。一旦细胞被隔离, 它们就可以用于各种实验技术, 如免疫组织化学、流式细胞术、蛋白质和 RNA 提取、随后的白细胞分离等. 试图了解不同细胞的作用次人口。通过使用实时 PCR 或基因组广泛的相关研究, 确定健康和复杂妊娠 (如先兆子痫或早产) 的蜕膜中基因和蛋白表达的差异, 可提供新的目标, 有助于诊断和治疗工具的开发。此外, 蜕细胞的比例, 特别是白细胞的变化, 可以很容易地识别通过使用流式细胞术, 也可以提供答案的病因的妊娠并发症, 如两个白细胞的变化亚人口比率以及它们的活化状态, 在足月和早产中被证明是可调的24。

子宫内膜是最活跃的成年女性组织之一, 通过对卵巢激素、雌激素 (E2) 和孕酮 (P4) 的刺激反应, 对每个月经周期进行戏剧性的重塑。蜕膜, 也称为怀孕子宫内膜, 是一个至关重要的生殖组织, 是由 postovulatory 阶段结束后形成的 P4-driven 分化后, E2-dominant 增生阶段。蜕细胞负责分泌的荷尔蒙因素, 成功的囊胚植入和发展的子宫-胎盘界面, 以保持产妇耐受胎儿移植。
蜕膜化是需要植入和随后重塑蜕螺旋动脉。子宫内膜基质细胞接受蜕膜化, 在 P4 和营地的控制下, 在月经周期的晚期黄体期1。这一过程是围绕血管和传播在整个基质, 表明其在血管重塑和白细胞贩运规则的作用。这种细胞转化的特点是圆形形态学, 增加核尺寸, 并扩大粗内质网和高尔基仪器2。Decidualized 基质细胞能够产生支持囊胚植入的分泌因子, 其特点是多种激素分泌 (即催乳素)、血管生成因子、胰岛素生长因子结合 protein-1(IGFBP-1), 前列腺素 (PG) E (细胞内的刺激器), 细胞因子, 细胞外基质成分和营养素为胎盘植入和发展所必需的3,4,5,6.
蜕细胞的数量不完全由 decidualized 基质细胞组成, 但也含有大量的、妊娠特异的蜕白细胞种群。蜕膜化包括短暂的局部水肿和自然杀伤细胞、T 细胞、树突状细胞和巨细胞的涌入。最大的白细胞亚群是子宫 NK 细胞, 包括大约50-70% 的所有母体白细胞浸润的蜕膜细胞因子和血管生成因子的来源, 这可能有助于蜕膜化过程中, 增加整个怀孕期间的数字7。巨噬细胞是免疫细胞的第二大亚群, 在植入部位周围发现并在妊娠期间增加8。它们是细胞因子和生长因子的来源, 如蜂群刺激因子 (CSF-1)9, 肿瘤坏死因子α (TNFα)10和前列腺素 (PG) E11。
在整个妊娠期和足月分娩之前, 蜕膜是细胞因子和趋化因子的主要来源, 负责孕产妇周围白细胞活化, 随后移入子宫组织以启动分娩。动物研究表明, 在分娩过程中, 老鼠蜕膜中有许多促炎性细胞因子被调节, 如 TNF a、IL-6、IL-12 和 IL-1b12。在人类蜕膜中, 促炎性细胞因子 IL-1b、IL-6 和 IL-8 (中性粒细胞趋化蛋白) 在分娩过程中表现出较高的表达, 而不是在劳动中13。这些分泌的细胞因子导致细胞的活化和流入蜕组织14;蜕巨噬细胞和中性粒细胞浸润在人和大鼠中的增加在足月劳动期间被看见, 与蜕入前浸润4倍更大, 表明在这两个相邻的子宫组织之间的激活级联15. 这些浸润白细胞产生的 PGs 能够激活子宫16, 基质金属蛋白酶 (基质) 的同步收缩, 以启动膜破裂17,18, 以及促炎性细胞因子放大子宫活化过程 ("细胞因子风暴")。
由于蜕细胞的许多重要功能, 如在植入过程中发挥关键作用, 在早期妊娠中保持孕产妇耐受性和参与分娩的激活, 不同的病理可能出现在妊娠。例如, (1) 因反复植入失败和复发性妊娠丢失而导致的不孕, 可能是蜕成熟失败所致;(2) 宫内生长限制 (IUGR) 和先兆子痫, 因发育不良和蜕膜/胎盘功能障碍或蜕-浸润交界的血管改变而受损;以及 (3) 早产可能是由于过早蜕激活所致。
鉴于这些主要疾病, 加上人类体内研究的伦理和实际局限性, 建立主要的人蜕细胞系对于体外分析是必不可少的, 目的是更好地了解和改善妊娠并发症的临床处理。因此, 我们的研究目的是制定一项协议, 允许隔离的人原蜕细胞高细胞产量和生存能力从胎儿细胞膜的足月胎盘。本协议明确描述了一种时间和经济高效的方法, 用于隔离用于各种体外分析的蜕细胞的特定子类型。蜕亚群的丰度和表型特征在足月和与第一或第二次妊娠的比较中是确定其在整个人类妊娠中的作用的关键。

<table><tbody><tr><td>Hank&amp;rsquo;s balanced salt solution with calcium and magnesium</td><td>Prepared in facility (LTRI)</td><td></td><td></td></tr><tr><td>Hank&amp;rsquo;s balanced salt solution without calcium and magnesium</td><td>Prepared in facility (LTRI)</td><td></td><td></td></tr><tr><td>Diaper pads</td><td>Sigma-Aldrich</td><td>D9542</td><td></td></tr><tr><td>Large surgical scissors</td><td>AL Medical</td><td>2018-12-20.</td><td></td></tr><tr><td>Large surgical forceps</td><td>Fine Science Tools</td><td>11000-18</td><td></td></tr><tr><td>Plastic disposable cell scraper (25 cm)</td><td>Sarstedt</td><td>83.183</td><td></td></tr><tr><td>250 mm (size 60 mesh) metal sieve</td><td>Sigma-Aldrich</td><td>S1020-5EA</td><td></td></tr><tr><td>Disposable scalpel with plastic handle (#21)</td><td>Fisher Scientific</td><td>08-927-5D</td><td></td></tr><tr><td>Sterile plastic petri dish (diameter 10 cm)</td><td>Sarstedt</td><td>82.1473.001</td><td></td></tr><tr><td>Sterile specimen container (urine cup, 4.5 oz)</td><td>VWR</td><td>25384-146</td><td></td></tr><tr><td>Nylon filter (70 mm)</td><td>VWR/Corning</td><td>21008-952</td><td></td></tr><tr><td>Erythrocyte lysis buffer</td><td>Qiagen</td><td>79217</td><td></td></tr><tr><td>Trypan blue, 0.4% solution</td><td>Lonza</td><td>17-942E</td><td></td></tr><tr><td>Parafilm</td><td>Fisher Scientific</td><td>13-374-10</td><td></td></tr><tr><td>Hemocytometer</td><td>Reichert</td><td>1490</td><td></td></tr><tr><td>Roswell Park Memorial Institute (RPMI) 1640 culture media</td><td>Invitrogen</td><td>11835-055</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Wisent</td><td>080-150</td><td></td></tr><tr><td>Normocin (50mg/ mL)</td><td>Invivogen</td><td>ant-nr-1</td><td></td></tr><tr><td>Plastic top filtration unit (0.22 mm membrane, 500 mL)</td><td>Millipore</td><td>SCGPT05RE</td><td></td></tr><tr><td>Collagenase 2, lyophilized powder</td><td>Sigma-Aldrich</td><td>C6885</td><td></td></tr><tr><td>Soy bean trypsin inhibitor, powder</td><td>Sigma-Aldrich</td><td>T9003-250mg</td><td></td></tr><tr><td>DNase powder</td><td>Roche</td><td>10104159001</td><td></td></tr><tr><td>Bovine serum albumin (BSA powder)</td><td>Fisher Scientific</td><td>BP1600-100</td><td></td></tr><tr><td>Spinning disc confocal microscope - Leica DMI 6000B</td><td>Leica</td><td></td><td></td></tr><tr><td>Imaging Flow cytometer - Image Stream MK2</td><td>Amnis</td><td></td><td></td></tr><tr><td>IDEA software</td><td>Millipore Sigma</td><td></td><td></td></tr><tr><td>APC-conjugated Vimentin antibody</td><td>R&amp;amp;D Systems</td><td>IC2105A</td><td></td></tr><tr><td>APC H7-conjugated CD45 antibody</td><td>BD</td><td>641399</td><td></td></tr><tr><td>FITC-conjugated Cytokeratin antibody</td><td>MACs Miltenyi Biotec</td><td>130-080-101</td><td></td></tr><tr><td>PerCP -conjugated Cytokeratin 7 antibody</td><td>Novus</td><td>NBP2-47941PCP</td><td></td></tr><tr><td>eFluor450 Fixable Viability dye</td><td>Thermo Fisher Scientific</td><td>65-0863-14</td><td></td></tr><tr><td>Vimentin primary antibody</td><td>Santa Cruz</td><td>sc-7558</td><td></td></tr><tr><td>CD45 primary antibody</td><td>Dako</td><td>M0701</td><td></td></tr><tr><td>Cytokeratin primary antibody</td><td>Dako</td><td>M0821</td><td></td></tr><tr><td>Cytokeratin 7 primary antibody</td><td>Dako</td><td>M7018</td><td></td></tr><tr><td>Mouse IgG</td><td>Santa Cruz</td><td>sc-2025</td><td></td></tr><tr><td>Goat IgG</td><td>Santa Cruz</td><td>sc-2028</td><td></td></tr><tr><td>Alexa Fluor 546 secondary antibody</td><td>Invitrogen</td><td>A10036</td><td></td></tr><tr><td>Alexa Fluor 594 secondary antibody</td><td>Fisher Scientific</td><td>A-11058</td><td></td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9542</td><td></td></tr></tbody></table>

isolation of primary human decidual cells from the fetal membranes of term placentae

蜕膜, 也称为怀孕子宫内膜, 是至关重要的生殖组织。蜕细胞主要由 decidualized 基质细胞和免疫细胞组成, 负责分泌激素和炎症因子, 这些因素对于成功的囊胚植入、胎盘发育和在在足月和早产时开始分娩。许多妊娠并发症可能是由不同细胞种群的细微平衡的扰动引起的, 包括蜕膜。改变特定蜕细胞类型的比例可能会扰乱这些关键的过程, 并增加发生严重妊娠并发症的风险, 如胚胎植入失败, 宫内生长限制, 先兆子痫和早产。此处概述的协议说明了一种成本和时间有效的方法, 以隔离从足月胎盘的胎儿细胞膜收集的主要人类蜕细胞。结合酶消化和蜕组织的温和机械破坏, 获得了蜕细胞的高产, 几乎没有绒毛膜污染。重要的是, 分离的蜕细胞的特征 (基质细胞 (55-60%), 白细胞 (35%), 上皮 (1%) 或滋养层 (0.01%) 细胞) 和维持高生存能力 (80%), 这是由多色成像流式细胞仪检测证实。这个协议是特定于蜕膜颞骨, 可以适应第一和第二个月胎盘。一旦分离, 蜕细胞可用于众多的实验应用, 目的是了解不同蜕细胞亚群在妊娠并发症中的作用。

为验证分离细胞的有效性和生存能力, 采用流式细胞术和免疫组织化学 (ICC) 两种方法进行了表征。4个细胞数量被瞄准了;用抗波形抗体检测 decidualized 基质细胞, 用 pan 白细胞标记 CD45 识别蜕免疫细胞, 用角蛋白检测上皮/内皮细胞, 最后用角蛋白7检测任何潜在滋养层 (绒毛膜或胎盘) 污染。
对于多色成像流式细胞仪的表征, 新鲜分离的蜕细胞用一种可修复的染料染色来确定细胞的生存能力。细胞透和固定, 其次是染色 fluorochrome 共轭小鼠单克隆抗体靶向波形 APC, CD45-APC-H7, 角蛋白 FITC 和角蛋白 7-PerCP。抗体信息列在材料表中。固定和染色的细胞储存在4°c 过夜的染色缓冲, 以防止串联染料的离解和运行次日在图像流 MK2 成像流式细胞仪。使用软件进行分析。使用三序贯特征/掩码组合 (图 1A-b) 的串行浇口排除了单元碎片和聚合体。荧光减去一 (FMO) 浇口控制用于识别阳性蜕细胞群和单一染色细胞用于补偿 (图 2)。最后的点图显示了四标记 (图 2) 的阳性染色细胞数量和 80% (图 1C) 的细胞生存能力。在图 1D中可以看到具有阳性染色细胞的代表性图像。利用这种方法, 我们发现, 种群主要由基质细胞 (55-60%)、白细胞 (35%)、上皮 (1%) 或滋养层 (0.01%) 细胞组成 (图 1E)。实验重复了三次。这些结果验证了纯度 (没有绒毛膜滋养层的污染) 和原始人类蜕细胞的高生存能力, 收集和分离的胚胎膜的足月胎盘。
在一个独立的 ICC 实验中, 新鲜分离的蜕细胞在玻璃盖玻片 (7.5万细胞/盖玻片) 上播种, 允许附着并保持在培养中48小时。介质在24小时后被改变以去除未附着的死细胞。然后用 50% acetone-50%methanol 固定, 透0.02% 非离子表面活性剂, 用阻断溶液阻断非特异性束缚。然后用小鼠单克隆抗体对细胞进行染色: anti-CD45 (pan 白细胞标记), 抗角蛋白 (上皮细胞标记) 和抗角蛋白 7 (滋养层标记), 山羊多克隆抗波形抗体 (基质细胞标记)。驴抗山羊和驴抗鼠被用作二级抗体。DAPI 被用来染色细胞核。小鼠 igg、山羊 igg 和二次抗体单独被用作阴性和特异性控制。在旋转圆盘共焦显微镜上, 以20X 倍的比例拍摄图像。使用的抗体列在材料表中, 在图 3中显示有代表性的图像。我们的视觉观察表明, 大多数附着的主蜕细胞是波形阳性基质细胞 (约 95%), 与少量的白细胞 (约 1-2%), 上皮 (约 1%) 和滋养细胞的人群 (单个细胞检测 < 0.01% 的会计)。流量细胞与 ICC 结果的主要区别是流式细胞术检测 CD45 阳性白细胞数量的减少。这一差异可以用两个实验的方法来解释: 在流式细胞术分析中, 孤立的蜕细胞被立即处理和染色, 这意味着在分析中包括白细胞的数量。与 ICC, 总蜕细胞悬浮被镀和培养48小时在媒介, 主要支持连接的基质和上皮细胞, 但不是免疫细胞。在24小时之后, 没有附加的免疫细胞被移除在媒介变化期间, 计算结果的区别由这两种方法提出。我们的结论是, 研究复杂妊娠的人蜕膜中白细胞亚群应采用特别预防措施, 以避免意外消除白细胞数量。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57443/57443fig1.jpg"> 图 1: 蜕细胞群体歧视的门控策略。(A)刚隔离的固定蜕单元最初是封闭的, 以排除单元格双峰, (B)随后使用三顺序特征/掩码组合来排除细胞碎片。(C)活细胞 (粉红色门, 占总人口的 80%) 与死细胞 (蓝门, 总人口的 20%) 区别使用一个固定的生存能力染料。(D)具有生存能力标记和四荧光共轭抗体 (Ab) (波形 APC、CD45-APC-H7、角蛋白-FITC 和角蛋白 7-PerCP) 的蜕细胞的代表性图像。虚线刻度条等于7微米。(E)一旦确定了无碎片的活细胞种群, 这些标记用于确定蜕细胞群中每个细胞类型的百分比。对于图 1B和1C, 每个点表示单个蜕单元格。对于图 1A和1C, 斑点图的颜色范围描述了细胞密度, 红色表示高度稠密的细胞和蓝色密度较低的细胞。这里提供的数据代表了3种生物复制。<a href="/files/ftp_upload/57443/57443fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57443/57443fig2.jpg"> 图 2: Representativeflow 细胞分析主要的人类蜕细胞与 FMO 浇块.有代表性的彩色斑点图细胞从期限蜕膜和染色与(A)波形 APC (基质细胞标记), (B) CD45-APC-H7 (白细胞标记), (C) 角蛋白-FITC (上皮细胞标记) 和(D)角蛋白 7-PerCP (滋养层标记) 在各自 FMO 控制旁边。在流式细胞仪数据分析中, 采用荧光-减一 (FMO) 控制, 含有完全抗体鸡尾酒减去所讨论的标记并与完全染色的实验样品相同, 以确定真正的阳性蜕细胞的数量。FMO 控件显示相对背景, 并允许准确地对真实正信号进行门控。这里提供的数据代表了3种生物复制。<a href="/files/ftp_upload/57443/57443fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57443/57443fig3.jpg"> 图 3: 主要人类蜕细胞的代表性免疫化学分析.主要的人蜕细胞生长在玻璃盖玻片48小时, 随着培养基的变化, 24 小时后, 细胞被固定与丙酮甲醇和染色的单克隆小鼠 anti-CD45 (白细胞标记), 抗角蛋白 (上皮细胞标记) 和抗角蛋白 7 (滋养层标记) 抗体, 山羊多克隆抗波形抗体 (基质细胞标记)。代表性图像显示(A)波形阳性成纤维细胞 (次级 abs: 驴抗山羊), (B) CD45+ 白细胞 (二次 abs: 驴抗鼠), (C) 角蛋白阳性上皮细胞 (二次 abs: 驴抗鼠) 和(D)角蛋白7阳性滋养细胞 (二次 Abs: 驴抗鼠) 检测到 (红色染色)。DAPI 染色用于染色细胞细胞核 (蓝色染色)。非特异小鼠和山羊 igg (m igg 和 g igg) 被用作阴性对照, 省略主要 Abs 作为继发抗体控制 (m 2nd只和 G 2nd )。在 DMI 旋转圆盘共焦显微镜上, 图像以20X 的放大倍数拍摄。刻度条 = 32 微米。<a href="/files/ftp_upload/57443/57443fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae at JoVE.com

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

该协议展示了一种从足月胎盘的胎儿细胞膜中分离出的主要人类蜕细胞的方法, 可用于各种应用 (即免疫细胞化学、流式术中、等), 目的是研究不同细胞群在妊娠并发症中的作用。

足月胎盘胎膜中原发性人蜕细胞的分离

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

isolation-primary-human-decidual-cells-from-fetal-membranes-term

Nanyang Technological University

Research

JoVE Journal

Medicine

14.1K 次观看.  University of Toronto. 该协议展示了一种从足月胎盘的胎儿细胞膜中分离出的主要人类蜕细胞的方法, 可用于各种应用 (即免疫细胞化学、流式术中、等), 目的是研究不同细胞群在妊娠并发症中的作用。

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

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.

在新鲜分离的人代绒毛滋养细胞转染和EMSA分析

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

科研

遗传学

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

从人胎盘组织源分离和间充质干扩张/基质细胞

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

发育生物学

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.

人胎盘滋养层细胞培养模型，研究褪黑素对缺氧的保护作用/复氧引起的中断

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

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

从胎盘中的造血干细胞的分离和分析

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

生物学

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. Although many efforts have been made to reveal the underlying mechanisms of decidualization, the exact signaling between the epithelial cells that are in contact with the embryo and the underlying stromal cells remains poorly understood. Therefore, studying decidualization in a way that takes both the epithelial and stromal cells into account could improve our knowledge about the molecular details of decidualization. For this purpose, in vivo models of artificial decidualization are physiologically the most relevant; however, manipulation of intercellular communication is limited. Currently, in vitro cultures of endometrial stromal cells are being used to investigate the modulation of decidualization by several signaling molecules. Conventionally, human or mouse endometrial stromal cells are used. However, the availability of human samples is very often limited. Furthermore, the use of murine tissues is accompanied with variety in the method of culturing. This study presents a validated and standardized method to obtain pure Endometrial Epithelial Cell (EEC) and Stromal Cell (ESC) cultures using adult intact mice treated with estrogen for three consecutive days. The protocol is optimized to improve the yield, viability, and purity of the cells and was further extended in order to study decidualization in a coculture of EEC and ESC. This model may be suitable to exploit the importance of both cell types in decidualization and to evaluate the contribution of significant signaling molecules secreted by EEC or ESC during the intercellular communication.

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

This study presents a standardized and validated method for the isolation and culture of primary mouse endometrial stromal and epithelial cells, which can be used in a coculture system to study in vitro decidualization.

鼠标子宫内膜上皮细胞和基质细胞的隔离<em&gt;体外</em&gt;蜕膜

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. ...

Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of neutral lipids surrounded by a phospholipid monolayer. One of the most useful techniques in determining the cellular roles of droplets has been proteomic identification of bound proteins, which can be isolated along with the droplets. Here, two methods are described to isolate lipid droplets and their bound proteins from two wide-ranging eukaryotes: fission yeast and human placental villous cells. Although both techniques have differences, the main method - density gradient centrifugation -&#160;is shared by both preparations. This shows the wide applicability of the presented droplet isolation techniques.

In the first protocol, yeast cells are converted into spheroplasts by enzymatic digestion of their cell walls. The resulting spheroplasts are then gently lysed in a loose-fitting homogenizer. Ficoll is added to the lysate to provide a density gradient, and the mixture is centrifuged three times. After the first spin, the lipid droplets are localized to the white-colored floating layer of the centrifuge tubes along with the endoplasmic reticulum (ER), the plasma membrane, and vacuoles. Two subsequent spins are used to remove these other three organelles. The result is a layer that has only droplets and bound proteins.

In the second protocol, placental villous cells are isolated from human term placentas by enzymatic digestion with trypsin and DNase I. The cells are homogenized in a loose-fitting homogenizer. Low-speed and medium-speed centrifugation steps are used to remove unbroken cells, cellular debris, nuclei, and mitochondria. Sucrose is added to the homogenate to provide a density gradient and the mixture is centrifuged to separate the lipid droplets from the other cellular fractions.

The purity of the lipid droplets in both protocols is confirmed by Western Blot analysis. The droplet fractions from both preps are suitable for subsequent proteomic and lipidomic analysis.

Two techniques for isolating cellular lipid droplets from 1) yeast cells and 2) human placentas are presented. The centerpiece of both procedures is density gradient centrifugation, where the resulting floating layer containing the droplets can be readily visualized by eye, extracted, and quantified by Western Blot analysis for purity.

隔离细胞脂滴:两个纯化技术从酵母细胞与人类的胎盘开始

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of ...

生物工程

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for embryo implantation into the uterine lining of the maternal womb. Improper decidualization has been established as a root cause for implantation failure and subsequent early embryo miscarriage. Therefore, understanding the molecular mechanisms underlying decidualization is advantageous to improving the rate of successful births. In vivo based studies of artificial decidualization are often limiting due to ethical dilemmas associated with human research, as well as translational complications within animal models. As a result, in vitro assays through primary cell culture are often utilized to explore the modulation of decidualization via hormones. This study provides a detailed protocol for the isolation of HESC and subsequent artificial decidualization via the supplementation of hormones to the culturing medium. Further, this study provides a well-designed method to knockdown any gene of interest by utilizing lipid-based siRNA transfections. This protocol permits the optimization of culture purity as well as product yield, thereby maximizing the ability to utilize this model as a reliable method to understand the molecular mechanisms underlying decidualization, and the subsequent quantification of secreted agents by decidualized endometrial stromal cells.

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

This study presents a validated and optimized procedure for the isolation and culture of human endometrial stromal cells to conduct in vitro decidualization assay. Further, this study provides a detailed method to efficiently knockdown a specific gene using siRNAs in human endometrial stromal cells.

人子宫内膜基质细胞的体外分离蜕膜化

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for ...

Isolation of Leukocytes from the Human Maternal-fetal Interface

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua parietalis). This interface is the anatomical site of contact between maternal and fetal tissues; therefore, it is an immunological site of action during pregnancy. Infiltrating leukocytes at the maternal-fetal interface play a central role in implantation, pregnancy maintenance, and timing of delivery. Therefore, phenotypic and functional characterizations of these leukocytes will provide insight into the mechanisms that lead to&#160;pregnancy disorders. Several protocols have been described in order to isolate infiltrating leukocytes from the decidua basalis and decidua parietalis; however, the lack of consistency in the reagents, enzymes, and times of incubation makes it difficult to compare these results. Described herein is&#160;a novel approach that combines the use of gentle mechanical and enzymatic dissociation techniques to preserve the viability and integrity of extracellular and intracellular markers in leukocytes isolated from the human tissues at the maternal-fetal interface. Aside from immunophenotyping, cell culture, and cell sorting, the future applications of this protocol are numerous and varied. Following this protocol, the isolated leukocytes can be used to determine DNA methylation, expression of target genes, in vitro leukocyte functionality (i.e., phagocytosis, cytotoxicity, T-cell proliferation, and plasticity, etc.), and the production of reactive oxygen species at the maternal-fetal interface. Additionally, using the described protocol, this laboratory has been able to describe new and rare leukocytes at the maternal-fetal interface.

Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis - the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis.

从人类母胎界面分离白细胞

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua ...

免疫与感染

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

分离，冻存和文化的人羊膜上皮细胞的临床应用

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

医学

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

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 in placental development have important consequences for the fetus and mother, and are a major cause of embryonic lethality. The major cell type of the 
fetal portion of the placenta is the trophoblast. Primary mouse placental trophoblast cells are a useful tool for studying normal and abnormal placental development, 
and unlike cell lines, may be isolated and used to study trophoblast at specific stages of pregnancy. In addition, primary cultures of trophoblast from transgenic
mice may be used to study the role of particular genes in placental cells. The protocol presented here is based on the description by Thordarson et al.1, in which a 
percoll gradient is used to obtain a relatively pure trophoblast cell population from isolated mouse placentas. It is similar to the more widely used methods for 
human trophoblast cell isolation2-3. Purity may be assessed by immunocytochemical staining of the isolated cells for cytokeratin 74. Here, the isolated cells are 
then analyzed using a matrigel invasion assay to assess trophoblast invasiveness in vitro5-6. The invaded cells are analyzed by immunocytochemistry and stained 
for counting.

In this protocol, we describe the dissection of placentae from the mouse on pregnancy d10.5, followed by isolation of trophoblast cells using a Percoll gradient. We then demonstrate use of the isolated cells in a matrigel invasion assay.

分离小鼠原代滋养细胞及滋养细胞侵袭实验

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 ...