Name: in vitro culture of epithelial cells from different anatomical regions of the human amniotic membrane
Uploaded: 2019-11-28T14:00:00
Description: Watch this Scientific Journal Video about In Vitro Culture of Epithelial Cells from Different Anatomical Regions of the Human Amniotic Membrane at JoVE.com

我们的研究得到了墨西哥国家研究所（21041年和21081年）和国家委员会（A1-S-8450和252756）的资助。我们感谢杰西卡·冈萨雷斯·诺里斯和莉迪亚·尤里娅·帕里德斯·维维斯的技术支持。

该协议由墨西哥城国家研究所伦理委员会批准（注册号212250-21041）。这些研究中进行的所有程序均符合《国家佩里纳洛尼亚研究所》、《赫尔辛基宣言》和卫生部《墨西哥官方标准》中规定的指导方针的道德标准。
1. 准备
<ol><li>使用 EDTA 准备 1x PBS 解决方案。为此，将 500 μL 的 0.5 M EDTA 库存添加到 500 mL 的 1x PBS 中，最终浓度为 0.5 mM EDTA。</li>
	<li>为 HAEC 准备培养基。服用450 ...

<ol>
	<li>Shahbazi, M. N., 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=Self-organization+of+the+human+embryo+in+the+absence+of+maternal+tissues.">Self-organization of the human embryo in the absence of maternal tissues.</a> Nature Cell Biology. 18 (6), 700-708 (2016).</li><li>Garcia-Lopez, 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=Human+amniotic+epithelium+(HAE)+as+a+possible+source+of+stem+cells+(SC).">Human amniotic epithelium (HAE) as a possible source of stem cells (SC).</a> Gaceta Medica de Mexico. 151 (1), 66-74 (2015).</li><li>Gramignoli, R., Srinivasan, R. C., Kannisto, K., Strom, 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=Isolation+of+Human+Amnion+Epithelial+Cells+According+to+Current+Good+Manufacturing+Procedures.">Isolation of Human Amnion Epithelial Cells According to Current Good Manufacturing Procedures.</a> Current Protocols in Stem Cell Biology. 37, (2016).</li><li>Murphy, 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=Amnion+epithelial+cell+isolation+and+characterization+for+clinical+use.">Amnion epithelial cell isolation and characterization for clinical use.</a> Current Protocols in Stem Cell Biology. , (2010).</li><li>Garcia-Castro, I. 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=Markers+of+Pluripotency+in+Human+Amniotic+Epithelial+Cells+and+Their+Differentiation+to+Progenitor+of+Cortical+Neurons.">Markers of Pluripotency in Human Amniotic Epithelial Cells and Their Differentiation to Progenitor of Cortical Neurons.</a> PLoS One. 10 (12), 0146082 (2015).</li><li>Garcia-Lopez, 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=Pluripotency+markers+in+tissue+and+cultivated+cells+in+vitro+of+different+regions+of+human+amniotic+epithelium.">Pluripotency markers in tissue and cultivated cells in vitro of different regions of human amniotic epithelium.</a> Experimental Cell Research. 375 (1), 31-41 (2019).</li><li>Yang, P. 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=Biological+characterization+of+human+amniotic+epithelial+cells+in+a+serum-free+system+and+their+safety+evaluation.">Biological characterization of human amniotic epithelial cells in a serum-free system and their safety evaluation.</a> Acta Pharmacological Sinica. 39 (8), 1305-1316 (2018).</li><li>Zou, 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=MicroRNA32+silences+WWP2+expression+to+maintain+the+pluripotency+of+human+amniotic+epithelial+stem+cells+and+beta+isletlike+cell+differentiation.">MicroRNA32 silences WWP2 expression to maintain the pluripotency of human amniotic epithelial stem cells and beta isletlike cell differentiation.</a> International Journal of Molecular Medicine. 41 (4), 1983-1991 (2018).</li><li>Avila-Gonzalez, D., 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=Capturing+the+ephemeral+human+pluripotent+state.">Capturing the ephemeral human pluripotent state.</a> Developmental Dynamics. 245 (7), 762-773 (2016).</li><li>Niknejad, H., 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=Properties+of+the+amniotic+membrane+for+potential+use+in+tissue+engineering.">Properties of the amniotic membrane for potential use in tissue engineering.</a> European Cells &amp; Materials. 15, 88-99 (2008).</li><li>Miki, T. <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+characteristics+and+the+therapeutic+potential+of+amniotic+epithelial+cells.">Stem cell characteristics and the therapeutic potential of amniotic epithelial cells.</a> American Journal of Reproductive Immunology. 80 (4), 13003 (2018).</li><li>Wu, Q., 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=Comparison+of+the+proliferation,+migration+and+angiogenic+properties+of+human+amniotic+epithelial+and+mesenchymal+stem+cells+and+their+effects+on+endothelial+cells.">Comparison of the proliferation, migration and angiogenic properties of human amniotic epithelial and mesenchymal stem cells and their effects on endothelial cells.</a> International Journal of Molecular Medicine. 39 (4), 918-926 (2017).</li><li>Hammer, A., 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=Amnion+epithelial+cells,+in+contrast+to+trophoblast+cells,+express+all+classical+HLA+class+I+molecules+together+with+HLA-G.">Amnion epithelial cells, in contrast to trophoblast cells, express all classical HLA class I molecules together with HLA-G.</a> American Journal of Reproductive Immunology. 37 (2), 161-171 (1997).</li><li>Benirschke, K., 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=Anatomy+and+Pathology+of+the+Placental+Membranes.">Anatomy and Pathology of the Placental Membranes.</a> Pathology of the Human Placenta. , 268-318 (1995).</li><li>Banerjee, A., 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=Different+metabolic+activity+in+placental+and+reflected+regions+of+the+human+amniotic+membrane.">Different metabolic activity in placental and reflected regions of the human amniotic membrane.</a> Placenta. 36 (11), 1329-1332 (2015).</li><li>Kim, S. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-143+regulation+of+prostaglandin-endoperoxidase+synthase+2+in+the+amnion:+implications+for+human+parturition+at+term.">miR-143 regulation of prostaglandin-endoperoxidase synthase 2 in the amnion: implications for human parturition at term.</a> PLoS One. 6 (9), 24131 (2011).</li><li>Han, Y. 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=Region-specific+gene+expression+profiling:+novel+evidence+for+biological+heterogeneity+of+the+human+amnion.">Region-specific gene expression profiling: novel evidence for biological heterogeneity of the human amnion.</a> Biology of Reproduction. 79 (5), 954-961 (2008).</li><li>Alcaraz, A., 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=Autocrine+TGF-beta+induces+epithelial+to+mesenchymal+transition+in+human+amniotic+epithelial+cells.">Autocrine TGF-beta induces epithelial to mesenchymal transition in human amniotic epithelial cells.</a> Cell Transplantation. 22 (8), 1351-1367 (2013).</li><li>Canciello, A., 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=Progesterone+prevents+epithelial-mesenchymal+transition+of+ovine+amniotic+epithelial+cells+and+enhances+their+immunomodulatory+properties.">Progesterone prevents epithelial-mesenchymal transition of ovine amniotic epithelial cells and enhances their immunomodulatory properties.</a> Scientific Reports. 7 (1), 3761 (2017).</li><li>Canciello, A., Greco, L., Russo, V., Barboni, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amniotic+Epithelial+Cell+Culture.">Amniotic Epithelial Cell Culture.</a> Methods in Molecular Biology. 1817, 67-78 (2018).</li><li>Singh, A. 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=Signaling+network+crosstalk+in+human+pluripotent+cells:+a+Smad2/3-regulated+switch+that+controls+the+balance+between+self-renewal+and+differentiation.">Signaling network crosstalk in human pluripotent cells: a Smad2/3-regulated switch that controls the balance between self-renewal and differentiation.</a> Cell Stem Cell. 10 (3), 312-326 (2012).</li><li>Villa-Diaz, L. G., Kim, J. K., Laperle, A., Palecek, S. P., Krebsbach, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+Focal+Adhesion+Kinase+Signaling+by+Integrin+alpha6beta1+Supports+Human+Pluripotent+Stem+Cell+Self-Renewal.">Inhibition of Focal Adhesion Kinase Signaling by Integrin alpha6beta1 Supports Human Pluripotent Stem Cell Self-Renewal.</a> Stem Cells. 34 (7), 1753-1764 (2016).</li><li>Bednar, A. D., Beardall, M. K., Brace, R. A., Cheung, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+and+regional+distribution+of+aquaporins+in+amnion+of+normal+and+gestational+diabetic+pregnancies.">Differential expression and regional distribution of aquaporins in amnion of normal and gestational diabetic pregnancies.</a> Physiological Reports. 3 (3), 12320 (2015).</li><li>Avila-Gonzalez, D., 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=Human+amniotic+epithelial+cells+as+feeder+layer+to+derive+and+maintain+human+embryonic+stem+cells+from+poor-quality+embryos.">Human amniotic epithelial cells as feeder layer to derive and maintain human embryonic stem cells from poor-quality embryos.</a> Stem Cell Research. 15 (2), 322-324 (2015).</li></ol>

我们实现了一种新的协议，将HAEC与术语膜分离开来。它不同于以前的报告，因为每个膜在分离前被分成三个解剖区域，以分析每个膜的细胞。
该协议中最关键的步骤之一是清洗膜以去除所有血块，因为它们在分离上皮细胞时会干扰胰蛋白酶的活动。未能正确执行此步骤可能导致获得具有过量红细胞和很少粘附上皮细胞的主要培养物。如果在设定细胞种子后的前 24-48 小时未观...

人类羊皮上皮细胞（HAEC）起源于胚胎发育的早期阶段，在受精后8天左右。它们产生于从羊膜1的最内层衍生的表征细胞的鳞状上皮细胞。因此，HAEC被认为是表征中多能细胞的残余物，有可能分化成胚胎2的三个生殖层。在过去的十年中，不同的研究小组已经开发出了在妊娠期内将这些细胞从羊膜分离出来的方法，以在体外3、4的培养模型中描述其推定多能相关特性。
因此，已发现HAEC具有人类多能干细胞（HPSC）的特征特征，如表面抗原SSEA-3、SSEA-4、TRA 1-60、TRA 1-81;多能转录因子OCT4、SOX2和NANOG的核心;和扩散标记KI67，表明他们是自我更新5，6，7。此外，这些细胞已经受到挑战，使用分化协议获得细胞阳性的生殖层（外生，中生代和内皮）4，5，8，以及人类疾病的动物模型。最后，HAEC表示E-cadherin，这表明他们保留了上皮性质很像HPSC5，9。
除了胚胎来源外，HAEC还有其他内在特性，使它们适合不同的临床应用，如抗炎和抗菌分子的分泌10，11，生长因子和细胞因子释放12，不形成畸马细胞瘤时，他们移植到免疫缺陷小鼠与HPSC2对比，和免疫耐受性，因为它们表达HLA-G，减少排斥后的风险移植13.
然而，以前的报告假设人类羊膜是同质膜，没有考虑到它可以在解剖学和生理上分为三个区域：胎盘（覆盖去皮底的羊膜），脐带（包围脐带的部分），并反射（膜的其余部分不附着在胎盘上）14 。研究表明，羊驼的胎盘和反射区域在形态学、线粒体活性、活性氧物种15检测、miRNA表达16和信号通路17激活方面存在差异。这些结果表明，人类羊皮被一个具有不同功能的异质人群所整合，在原位或体外模型中进行进一步的研究应考虑。虽然其他实验室已经设计了从整个膜分离HAEC的协议，但我们的实验室已经建立了一个协议来分离、培养和表征来自不同解剖区域的细胞。

<table>
	<tbody>
		<tr>
			<td>Culture reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Thermo Fisher Scientific/Gibco</td>
			<td>21985023</td>
			<td>55 mM</td>
		</tr>
		<tr>
			<td>Animal-Free Recombinant Human EGF</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>Thermo Fisher Scientific/Gibco</td>
			<td>15240062</td>
			<td>100X</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Thermo Fisher Scientific/Gibco</td>
			<td>12430054</td>
			<td>Supplemented with high glucose and HEPES</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Thermo Fisher Scientific/Ambion</td>
			<td>AM9260G</td>
			<td>0.5 M</td>
		</tr>
		<tr>
			<td>Embryonic stem-cell FBS, qualified</td>
			<td>Thermo Fisher Scientific/Gibco</td>
			<td>10439024</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-Essential Amino Acids</td>
			<td>Thermo Fisher Scientific/Gibco</td>
			<td>11140050</td>
			<td>100X</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>any brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline</td>
			<td>Thermo Fisher Scientific/Gibco</td>
			<td>10010023</td>
			<td>1X</td>
		</tr>
		<tr>
			<td>Saline solution (sodium chloride 0.9%)</td>
			<td>any brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Thermo Fisher Scientific/Gibco</td>
			<td>11360070</td>
			<td>100 mM</td>
		</tr>
		<tr>
			<td>Trypsin/EDTA 0.05%</td>
			<td>Thermo Fisher Scientific/Gibco</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#181;m Cell Strainer</td>
			<td>Corning/Falcon</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm TC-Treated Culture Dish</td>
			<td>Corning</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well Clear TC-treated Multiple Well Plates</td>
			<td>Corning/Costar</td>
			<td>3526</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well Clear TC-treated Multiple Well Plates</td>
			<td>Corning/Costar</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-Pyrogenic Sterile Centrifuge Tube</td>
			<td>any brand</td>
			<td></td>
			<td>with conical bottom</td>
		</tr>
		<tr>
			<td>Non-Pyrogenic sterile tips of 1,000 &#181;l, 200 &#181;l and 10 &#181;l.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile cotton gauzes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile serological pipettes of 5, 10 and 25 mL</td>
			<td>any brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile surgical gloves</td>
			<td>any brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biological safety cabinet</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipettes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Motorized Pipet Filler/Dispenser</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile beakers of 500 mL</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile plastic cutting board</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile scalpels, scissors, forceps, clamps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile stainless steel container</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile tray</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tube Rotator</td>
			<td>MaCSmix</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies and Kits</td>
			<td></td>
			<td></td>
			<td>Antibody ID</td>
		</tr>
		<tr>
			<td>Anti-E-cadherin</td>
			<td>BD Biosciences</td>
			<td>610181</td>
			<td>RRID:AB_3975</td>
		</tr>
		<tr>
			<td>Anti-KI67</td>
			<td>Santa Cruz</td>
			<td>23900</td>
			<td>RRID:AB_627859)</td>
		</tr>
		<tr>
			<td>Anti-NANOG</td>
			<td>Peprotech</td>
			<td>500-P236</td>
			<td>RRID:AB_1268274</td>
		</tr>
		<tr>
			<td>Anti-OCT4</td>
			<td>Abcam</td>
			<td>ab19857</td>
			<td>RRID:AB_44517</td>
		</tr>
		<tr>
			<td>Anti-SOX2</td>
			<td>Millipore</td>
			<td>AB5603</td>
			<td>RRID:AB_2286686</td>
		</tr>
		<tr>
			<td>Anti-SSEA-4</td>
			<td>Cell Signaling</td>
			<td>4755</td>
			<td>RRID:AB_1264259</td>
		</tr>
		<tr>
			<td>Anti-TRA-1-60</td>
			<td>Cell Signaling</td>
			<td>4746</td>
			<td>RRID:AB_2119059</td>
		</tr>
		<tr>
			<td>Goat Anti-Mouse Alexa Fluor 488</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-11029</td>
			<td>RRID:AB_2534088</td>
		</tr>
		<tr>
			<td>Goat Anti-Rabbit Alexa Fluor 568</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-11036</td>
			<td>RRID:AB_10563566</td>
		</tr>
		<tr>
			<td>Tunel Assay Kit</td>
			<td>Abcam</td>
			<td>66110</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro culture of epithelial cells from different anatomical regions of the human amniotic membrane

文献中报告了关于人类羊皮上皮细胞（HAEC）的隔离和培养的若干协议。然而，这些假设羊皮上皮是同质层。人类羊膜可分为三个解剖区域：反射、胎盘和脐带。每个区域有不同的生理作用，如在病理条件下。在这里，我们描述了一个协议，解剖人类羊羊组织在三个部分，并在体外维护它。在培养中，从反射羊膜中提取的细胞呈长方体形态，而来自胎盘和脐带的细胞是鳞状的。尽管如此，所有获得的细胞都有上皮表型，通过E-cadherin的免疫检测证明。因此，由于原位胎盘和反射区域在细胞成分和分子功能上有所不同，因此体外研究可能有必要考虑这些差异，因为它们可能对HAEC的使用产生生理影响。生物医学研究及这些细胞在再生医学中的可喜应用。

HAEC从羊膜的三个解剖区域分离出来，并在体外单独培养。在培养48小时后，具有上皮表型的细胞附着在板表面，尽管介质中也含有细胞碎片和浮细胞，一旦介质改变，这些细胞被移除（图3）。
在初级培养物（通道零，P0）的处理过程中，可能会出现一些并发症，从而干扰实验数据分析（图4）：在识别试剂污染或分离过程中存在细?...

Watch this Scientific Journal Video about In Vitro Culture of Epithelial Cells from Different Anatomical Regions of the Human Amniotic Membrane at JoVE.com

In Vitro Culture of Epithelial Cells from Different Anatomical Regions of the Human Amniotic Membrane

该协议描述了从人类羊膜的不同解剖区域分离上皮细胞，以确定其异质性和功能特性，以便可能应用于临床和物理病理学模型。

人类羊膜不同解剖区域的皮下细胞体外培养

Several protocols have been reported in the literature for the isolation and culture of human amniotic epithelial cells (HAEC). However, these assume that ...

该协议可以帮助回答有关从羊膜分离的人类羊膜上皮细胞的异质性和功能特性的关键问题。虽然以前的协议将细胞从共羊膜中分离出来，而不在区域之间辨别，但我们的协议允许从三个不同的解剖区域分离细胞种群的培养。要按区域分离羊膜，在无菌条件下的生物安全柜中，识别包裹脐带的脐带、覆盖脱膜的胎盘膜和反射区域，这被认为是未附着在胎盘上的羊膜的其余部分。要解剖脐带区域，请使用解剖钳子来容纳覆盖胎盘和脐带交界处的脐带膜部分，并使用手术刀解剖脐带周围的区域，同时拉伸以将区域与胆汁分离。然后，将分离的组织沉积在有标签的烧杯中，并加注100毫升的盐水溶液。要解剖胎盘的 amnion 区域，请使用无菌棉纱布去除覆盖胎盘的胆小板/安尼翁表面的血块。并使用解剖钳来抓住胎盘和反射区域之间边界上的膜。使用手术刀沿着胎盘的周长切割，将胎盘与胎盘分离，小心不要从胎盘上切割任何血管。将分离的组织放在另一个标有 300 毫升盐水溶液的烧杯中。将未附着在胎盘上的其他羊膜与胆囊分离。然后，将反射区域放在另一个标有 300 毫升盐水溶液的烧杯中。要清洗膜，请使用钳子将其放在无菌表面上，以便能够丢弃每个组织容器中的盐水溶液。将膜放回容器中，向脐带添加 100 毫升新鲜盐水溶液，向地盘和反射区域添加 300 毫升新鲜盐水溶液。使用解剖钳来搅拌膜，以去除任何血液残留物，并将膜放在无菌表面上，以便能够丢弃盐水溶液。然后，洗膜至少两次，正如刚刚证明的，直到组织是半透明的。对于不同羊膜区域的酶消化，将反射区和地盘区域切成两个或三个片段。将碎片和脐带放入单独的50毫升离心管中。将20毫升0.5%的三叶辛-EDTA添加到每管反射和胎盘区域组织片段中，将5毫升0.5%的三叶辛-EDTA添加到含有脐带组织的管子中。轻轻摇动管子 30 秒。然后将膜放在无菌表面上，丢弃 trypsin-EDTA 溶液，代之以 30 毫升新鲜 0.5% trypsin-EDTA 的反射和胎盘区域组织片段和 15 毫升新鲜 0.5% trypsin-EDTA 的脐带区域组织。然后将管子放在培养箱内的旋转器中，在37摄氏度下以每分钟20次旋转的速度旋转40分钟。在孵育结束时，将整个细胞溶液体积转移到新的锥形管中。并将37摄氏度的人类 AE 细胞的体积增加两倍，以在冰上的每个管子中。消化原管中任何剩余的组织片段，如刚刚证明的新鲜0.5%的三辛-EDTA。在第二次消化结束时，使用一对解剖钳子固定 amnion 部分的一端和第二对解剖钳，沿剩余组织挤压，以去除在以前的潜伏期未完全剥离的任何上皮细胞。然后，取出膜，将第二个消化液转移到第二组离心管中。并且用2倍于冰上新鲜37摄氏度人类E细胞培养的体积来灭活消化酶。对于人类 AE 细胞分离，通过离心沉淀细胞。并重新在每个管中用10毫升的新鲜37摄氏度人类 AE 细胞介质重新暂停颗粒。将每对消化拉入一根管子中，并通过 100 微米滤株过滤悬浮液，以清除任何细胞外基质碎屑。数数后，将三个区域中每个区域的人类AE细胞在37摄氏度的人类AE细胞培养基中以每厘米的3倍10至第四细胞的三倍10至第四细胞的单独100毫米板，辅以每毫升10毫微克的人类表皮生长因子。然后，在加湿的培养箱中，在37摄氏度的高温条件下孵育培养，直到下游对兴趣进行分析。经过48小时的培养，具有上皮表型的人类 AE 细胞粘附在板的表面，而上经应物包含细胞碎片和漂浮细胞，一旦介质改变，这些细胞可以去除。建议在识别细菌污染、由于膜洗涤不足而导致的红细胞过多、缺乏或非粘结细胞或具有成纤维细胞形态的细胞时，丢弃培养物并处理另一膜。人类 AE 细胞形态取决于细胞的起源，因为反射区的细胞表现出一种立体形态，并在鹅卵石单层中生长，胎盘和脐带区域的细胞是扁平和鳞状的。对E-cadherin的免疫荧光表明，初级培养物和亚培养物保持其上皮表型，这表明没有其他细胞类型的污染。此外，这些细胞是可行的，它们表达的Ki-67增殖标记就证明了这一点。虽然来自 amnion 的亚细胞在形态和功能上不同，但多能因子核心的表达和存在在从胎盘和反射区域派生的人类 AE 细胞中不会改变。由于人类羊膜上皮细胞是多能干细胞的可能来源，我们可以通过一系列特定定义协议来挑战它们，以阐明每个区域的这些细胞是否具有不同的潜力。在开始协议之前，检查患者的病史，以确保组织没有感染的微生物特征。

Research

JoVE Journal

Developmental Biology

Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos

日本鹌鹑胚胎的卵黄囊膜主内胚上皮细胞培养

We established an endodermal epithelial cell culture model (EEC) for studying the function of certain enzymes and proteins in mediating nutrient ...

科研

发育生物学

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

的发展<em&gt;体外</em&gt;检测以确定间充质细胞将经历上皮 - 间质转化的收缩功能

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

从人多能干细胞使用高度可重复的小分子为基础的分化协议心肌细胞规模化生产

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

从小鼠羊水细胞多能干细胞使用转座子系统的生产

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

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

Isolation of Mouse Endometrial Epithelial and Stromal Cells for 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. ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

人多能干细胞对原始和最终造血祖细胞定向分化的研究

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Reprogramming Primary Amniotic Fluid and Membrane Cells to Pluripotency in Xeno-free Conditions

无异条件下多原代羊水和膜细胞重新编程

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

人胚胎或诱导多能干细胞对视网膜色素上皮细胞的快速定向分化

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Application of Aorta-gonad-mesonephros Explant Culture System in Developmental Hematopoiesis

主动脉-性腺-肾外植体培养体系在发育性造血中的应用

The limitation of using mouse embryos for hematopoiesis studies is the added inconvenience in operations, which is largely due to the intrauterine ...

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

老年人刷膜的体外模型对视网膜色素上皮细胞的培养

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic ...