Our research was supported by grants from Instituto Nacional de Perinatolog&#237;a de M&#233;xico (21041 and 21081) and CONACYT (A1-S-8450 and&#160;252756). We thank Jessica Gonz&#225;lez Norris and Lidia Yuriria Paredes Vivas for the technical support.

This protocol was approved by the ethical committee of Instituto Nacional de Perinatolog&#237;a in Mexico City (Registry number 212250-21041). All procedures performed in these studies were in accordance with the ethical standards of the Instituto Nacional de Perinatolog&#237;a, the Helsinki Declaration, and the guidelines set forth in the Ministry of Health&#8217;s Official Mexican Standard.
1. Preparation
<ol>
	<li>Prepare a solution of 1x PBS with EDTA. To do so, add 500 &#956;L of 0.5 M ...

<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>

We implemented a new protocol to isolate HAEC from term membranes. It differs from previous reports in that each membrane was divided into its three anatomical regions prior to isolation to analyze cells from each one.
One of the most critical steps in the protocol is the washing of the membrane to remove all blood clots, because they can interfere with the activity of trypsin when separating the epithelial cells. Failure to carry out this step properly can lead to obtaining a primary culture ...

Human amniotic epithelium&#160;cells&#160;(HAEC) originate during the early stages of embryonic development, at around eight days postfertilization. They arise from a population of squamous epithelial cells of the epiblast that derive from the innermost layer of the amniotic membrane1. Thus, HAEC are considered&#160;remnants of pluripotent cells from the epiblast that have the potential to differentiate into the three germ layers of the embryo2. In the last decade, diverse&#160;research groups have developed methods to isolate&#160;these cells from the amniotic membrane at the term of gestation to characterize their presumptive pluripotency-related properties in a culture model&#160;in vitro3,4.&#160;
Accordingly, it&#160;has been found that HAEC feature traits characteristic of human pluripotent stem cells (HPSC), such as the surface antigens SSEA-3, SSEA-4, TRA 1-60, TRA 1-81; the core of pluripotency transcription factors OCT4, SOX2, and NANOG; and the proliferation marker KI67, suggesting that they are self-renewing5,6,7. Moreover, these cells have been challenged using differentiation protocols to obtain cells positive for lineage-specific markers of the three germ layers (ectoderm, mesoderm, and endoderm)4,5,8, as well as in animal models of human diseases. Finally, HAEC express E-cadherin, which demonstrate that they retain an epithelial nature much like the HPSC5,9.
Apart from their embryonic origin, HAEC have other intrinsic properties that make them suitable for different clinical applications, such as the secretion of anti-inflammatory and antibacterial molecules10,11, growth factors and cytokine release12, no formation of teratomas when they are transplanted into immunodeficient mice in contrast with HPSC2, and immunological tolerance because they express HLA-G, which decreases the risk of rejection after transplantation13.
However, previous reports have assumed that the human amnion is a homogenous membrane, without considering that it can be anatomically and physiologically divided into three regions: placental (the amnion that covers the decidua basalis), umbilical (the part that envelops the umbilical cord), and reflected (the rest of the membrane not attached to the placenta)14. It has been shown that the placental and reflected regions of the amnion display differences in morphology, mitochondrial activity, detection of reactive oxygen species15, miRNA expression16, and activation of signaling pathways17. These results suggest that the human amnion is integrated by a heterogeneous population with different functionality that should be considered for further studies carried out in either in situ or in vitro models. While other laboratories have designed protocols for the isolation of HAEC from the whole membrane, our laboratory has established a protocol to isolate, culture, and characterize cells from different anatomical regions.

<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

Several protocols have been reported in the literature for the isolation and culture of human amniotic epithelial cells (HAEC). However, these assume that the amniotic epithelium is a homogeneous layer. The human amnion can be divided into three anatomical regions: reflected, placental, and umbilical. Each region has different physiological roles, such as in pathological conditions. Here, we describe a protocol to dissect human amnion tissue in three sections and maintain it in vitro. In culture, cells derived from the reflected amnion displayed a cuboidal morphology, while cells from both placental and umbilical regions were squamous. Nonetheless, all the cells obtained have an epithelial phenotype, demonstrated by the immunodetection of E-cadherin. Thus, because the placental and reflected regions in situ differ in cellular components and molecular functions, it may be necessary for in vitro studies to consider these differences, because they could have physiological implications for the use of HAEC in biomedical research and the promising application of these cells in regenerative medicine.

HAEC were isolated from each of the three anatomical regions of the amniotic membrane and individually cultured in vitro. After 48 h of culture, cells with an epithelial phenotype adhered to the surface of the plate, although the media also contained cell debris and floating cells, which were removed once the medium was changed (Figure 3).
During the processing of primary culture (passage zero, P0), some complications could arise that can interfere with the experi...

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

This protocol describes the isolation of epithelial cells from different anatomical regions of the human amniotic membrane to determine their heterogeneity and functional properties for possible application in clinical and physiopathological models.

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

This protocol can help answer key questions about heterogeneity and functional properties of human amniotic epithelial cells isolated from amniotic membrane. While previous protocols isolated cells from co-amniotic membrane without discerning between regions, our protocol allows the culture of cell population isolated from three distinct anatomical regions. To separate the amniotic membrane by region, in a biosafety cabinet under sterile conditions, identify the umbilical amnion enveloping the umbilical cord, the placental amnion covering the decidua basalis, and the reflected region, which is considered the rest of the amnion that is not attached to the placenta.To dissect the umbilical amnion region, use dissecting forceps to hold the portion of the amnion membrane that covers the junction of the placenta and the umbilical cord, and use a scalpel to dissect the region that surrounds the cord, while stretching to separate the region from the chorion. Then, deposit the separated tissue in a labeled beaker with 100 milliliters of saline solution. To dissect the placental amnion region, use a sterile cotton gauze to remove the blood clots from the surface of the chorion/amnion that covers the placenta.And use dissecting forceps to grasp the membrane on the border between the placenta and the reflected region. Use a scalpel to cut along the circumference of the placenta and separate the placental amnion from the chorion, being careful not to cut any vessels from the placenta. Place the separated tissue in another labeled beaker with 300 milliliters of saline solution.And separate the rest of the amniotic membrane that is not attached to the placenta from the chorion. Then, place the reflected region in another labeled beaker with 300 milliliters of saline solution. To wash the membranes, use tweezers to place them on a sterile surface to be able to discard the saline solution from each container of tissue.Place the membranes back in the container and add 100 milliliters of fresh saline solution to the umbilical region and 300 milliliters of fresh saline solution to the placental and reflected regions. Use dissecting forceps to stir the membranes to remove any blood residue and place the membranes on the sterile surface to be able to discard the saline solutions. Then, wash the membranes at least two more times, as just demonstrated, until the tissues are translucent.For enzymatic digestion of the different amniotic membrane regions, cut the reflected and placental regions into two or three fragments. And place the fragments and the umbilical region into individual 50-milliliter centrifuge tubes. Add 20 milliliters of 0.5%trypsin-EDTA to each tube of reflected and placental region tissue fragments and five milliliters of 0.5%trypsin-EDTA to the tube containing the umbilical region tissue.Shake the tubes gently for 30 seconds. Then place the membranes on a sterile surface, discarding the trypsin-EDTA solution and replace it with 30 milliliters of fresh 0.5%trypsin-EDTA for the reflected and placental region tissue fragments and 15 milliliters of fresh 0.5%Trypsin-EDTA for the umbilical region tissue. Then place the tubes in a rotator inside an incubator for a 40-minute rotation at 20 rotations per minute at 37 degrees Celsius.At the end of the incubation, transfer the entire volume of cell solution into new conical tubes. And add two times the volume of 37-degree-Celsius human AE cells medium to each tube on ice. Digest any leftover tissue fragments in the original tubes with fresh 0.5%trypsin-EDTA, as just demonstrated.At the end of the second digestion, use a pair of dissecting forceps to hold one end of the amnion portion and a second pair of dissecting forceps to squeeze along the remaining tissue to remove any rows of epithelial cells that did not completely peel off during the previous incubation periods. Then, remove the membranes to be able to transfer the second digestion solution to a second set of centrifuge tubes. And inactivate the digestion enzymes with two times the volume of fresh 37-degree-Celsius human AE cell medium on ice.For human AE cell isolation, sediment the cells by centrifugation. And resuspend the pellet in each tube with 10 milliliters of fresh 37-degree-Celsius human AE cell medium. Pull each pair of digestions into a single tube and filter the suspensions through 100-micrometer strainers to remove any extracellular matrix debris.After counting, seed the human AE cells from each of the three regions into individual 100-millimeter plates at a three times 10 to the fourth cells per centimeters squared concentration in 37-degree-Celsius human AE cell medium, supplemented with 10 nanograms per milliliter of human epidermal growth factor. Then, incubate the cultures at 37 degrees Celsius under normoxic conditions in a humidified incubator until the downstream analysis of interest. After 48 hours of culture, human AE cells with an epithelial phenotype adhere to the surface of the plate, while the supernatant contains cell debris and floating cells that can be removed once the medium is changed.It is advisable to discard the cultures and process another membrane upon identifying the presence of bacterial contamination, excessive erythrocytes due to insufficient washing of the membranes, deficiently or nonadherent cells, or cells with a fibroblast morphology. Human AE cell morphology depends on the origin of the cells, as cells from the reflected zone demonstrate a cuboidal morphology and grow in a cobbled monolayer, and cells from the placental and umbilical regions are flatter and squamous. Immunofluorescence against E-cadherin reveals that the primary cultures and subcultures maintain their epithelial phenotype, suggesting that there is no contamination of another cell type.In addition, the cells are viable, as evidenced by their expression of the Ki-67 proliferation marker. Although the subpopulations from the amnion differ in their morphology and function, the expression and presence of the core of the pluripotency factors does not change in human AE cells derived from the placental and reflected regions. Since human amniotic epithelial cells are a possible source of pluripotent stem cells, we can challenge them through a line of specific definition protocols to elucidate whether these cells from each region have different potentials.Previous to a started protocol, review the medical history of the patient to be sure that the tissue exhibits no microbiological characteristics of infection.

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JoVE Journal

Developmental Biology

7.7K vistas.  Instituto Nacional de Perinatología. Este protocolo puede ayudar a responder preguntas clave sobre la heterogeneidad y las propiedades funcionales de las células epiteliales amnióticas humanas aisladas de la membrana amniótica. Mientras que los protocolos anteriores aislan las células de la membrana co-amniótica sin discernir entre regiones, nuestro protocolo permite el cultivo de la población celular aislada de tres regiones anatómicas distintas. Para separar la membrana amniótica por región, en un gabinete de biosegur...