Name: isolation, cryopreservation and culture of human amnion epithelial cells for clinical applications
Uploaded: 2014-12-21T15:00:00
Description: Watch this Scientific Journal Video about Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications at JoVE.com

The authors acknowledge financial support from the Victorian Government&#8217;s Operational Infrastructure Support Program.

NOT: plasenta vadeli elektif sezaryen için bir tercih, tekiz gebeliklerde sağlıklı toplanan edilmelidir. Yazılı bilgilendirilmiş onam kendi plasenta toplanması için verilmelidir. Sizin ilgili insan araştırma etik kurul onayı tüm koleksiyon ve insan dokularının kullanılması gerekir. Amniyon Epitel Hücreleri 1. İzolasyon <ol style="margin-left: 40px;"><li> Bir sınıf II biyolojik güvenlik kabini içinde steril bir yüzeye yerleştirin plasentayı. </li><li> Steril Hanks ...

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	<li>Murphy, S. V., Wallace, E. M., Jenkin, G., Appasani, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Stem Cells and Regenerative Medicine. 1, 243-264 (2011).</li><li>Murphy, S. V., Atala, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amniotic+fluid+and+placental+membranes:+unexpected+sources+of+highly+multipotent+cells.">Amniotic fluid and placental membranes: unexpected sources of highly multipotent cells.</a> Semin. Reprod. Med. 31 (1), 62-68 (2013).</li><li>Parolini, O., 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=Concise+review:+isolation+and+characterization+of+cells+from+human+term+placenta:+outcome+of+the+first+international+Workshop+on+Placenta+Derived+Stem+Cells.">Concise review: isolation and characterization of cells from human term placenta: outcome of the first international Workshop on Placenta Derived Stem Cells.</a> Stem Cells. 26 (2), 300-311 (2008).</li><li>Lim, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preterm+human+amnion+epithelial+cells+have+limited+reparative+potential.">Preterm human amnion epithelial cells have limited reparative potential.</a> Placenta. 34 (6), 486-492 (2013).</li><li>Tamagawa, T., Ishiwata, I., Saito, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+characterization+of+a+pluripotent+stem+cell+line+derived+from+human+amniotic+membranes+and+initiation+of+germ+layers+in+vitro.">Establishment and characterization of a pluripotent stem cell line derived from human amniotic membranes and initiation of germ layers in vitro.</a> Hum Cell. 17 (3), 125-130 (2004).</li><li>Miki, T., Marongiu, F., Ellis, E., Strom, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+amniotic+epithelial+stem+cells.">Isolation of amniotic epithelial stem cells.</a> Curr Protoc Stem Cell Biol. 1, Unit 1E.3 (2007).</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> Curr Protoc Stem Cell Biol. 1, Unit 1E.6 (2010).</li><li>Ilancheran, 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=Stem+cells+derived+from+human+fetal+membranes+display+multilineage+differentiation+potential.">Stem cells derived from human fetal membranes display multilineage differentiation potential.</a> Biol Reprod. 77 (3), 577-588 (2007).</li><li>Fliniaux, I., Viallet, J. P., Dhouailly, D., Jahoda, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+amnion+epithelium+into+skin+and+hair+follicles.">Transformation of amnion epithelium into skin and hair follicles.</a> Differentiation. 72 (9-10), 558-565 (2004).</li><li>Miki, T., Lehmann, T., Cai, H., Stolz, D. B., 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=Stem+cell+characteristics+of+amniotic+epithelial+cells.">Stem cell characteristics of amniotic epithelial cells.</a> Stem Cells. 23 (10), 1549-1559 (2005).</li><li>Avila, M., Espana, M., Moreno, C., Pena, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+ocular+surface+with+heterologous+limbal+epithelium+and+amniotic+membrane+in+a+rabbit+model.">Reconstruction of ocular surface with heterologous limbal epithelium and amniotic membrane in a rabbit model.</a> Cornea. 20 (4), 414-420 (2001).</li><li>Sankar, V., Muthusamy, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+human+amniotic+epithelial+cell+transplantation+in+spinal+cord+injury+repair+research.">Role of human amniotic epithelial cell transplantation in spinal cord injury repair research.</a> Neuroscience. 118 (1), 11-17 (2003).</li><li>Yuge, I., 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=Transplanted+human+amniotic+epithelial+cells+express+connexin+26+and+Na-K-adenosine+triphosphatase+in+the+inner+ear.">Transplanted human amniotic epithelial cells express connexin 26 and Na-K-adenosine triphosphatase in the inner ear.</a> Transplantation. 77 (9), 1452-1454 (2004).</li><li>Li, 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=Immunosuppressive+factors+secreted+by+human+amniotic+epithelial+cells.">Immunosuppressive factors secreted by human amniotic epithelial cells.</a> Invest Ophthalmol Vis Sci. 46 (3), 900-907 (2005).</li><li>Liu, T., 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+ameliorate+behavioral+dysfunction+and+reduce+infarct+size+in+the+rat+middle+cerebral+artery+occlusion+model.">Human amniotic epithelial cells ameliorate behavioral dysfunction and reduce infarct size in the rat middle cerebral artery occlusion model.</a> Shock. 29 (5), 603-611 (2008).</li><li>Venkatachalam, 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=Novel+neurotrophic+factor+secreted+by+amniotic+epithelial+cells.">Novel neurotrophic factor secreted by amniotic epithelial cells.</a> Biocell. 33 (2), 81-89 (2009).</li><li>Manuelpillai, U., 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+cell+transplantation+induces+markers+of+alternative+macrophage+activation+and+reduces+established+hepatic+fibrosis.">Human amniotic epithelial cell transplantation induces markers of alternative macrophage activation and reduces established hepatic fibrosis.</a> PLoS One. 7 (6), e38631 (2012).</li><li>Wei, J. P., 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+amnion-isolated+cells+normalize+blood+glucose+in+streptozotocin-induced+diabetic+mice.">Human amnion-isolated cells normalize blood glucose in streptozotocin-induced diabetic mice.</a> Cell Transplant. 12 (5), 545-552 (2003).</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=Human+amnion+epithelial+cells+prevent+bleomycin-induced+lung+injury+and+preserve+lung+function.">Human amnion epithelial cells prevent bleomycin-induced lung injury and preserve lung function.</a> Cell Transplant. 20 (6), 909-923 (2011).</li><li>Murphy, S. V., 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+amnion+epithelial+cells+induced+to+express+functional+cystic+fibrosis+transmembrane+conductance+regulator.">Human amnion epithelial cells induced to express functional cystic fibrosis transmembrane conductance regulator.</a> PLoS One. 7 (9), e46533 (2012).</li><li>Murphy, S. V., 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+amnion+epithelial+cells+do+not+abrogate+pulmonary+fibrosis+in+mice+with+impaired+macrophage+function.">Human amnion epithelial cells do not abrogate pulmonary fibrosis in mice with impaired macrophage function.</a> Cell Transplant. 21 (7), 1477-1492 (2012).</li><li>Hodges, R. J., Lim, R., Jenkin, G., Wallace, E. M. <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+as+a+candidate+therapy+for+acute+and+chronic+lung+injury.">Amnion epithelial cells as a candidate therapy for acute and chronic lung injury.</a> Stem cells Int. 2012, 709763 (2012).</li><li>Tan, J. L., Chan, S. T., Wallace, E. M., Lim, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+amnion+epithelial+cells+mediate+lung+repair+by+directly+modulating+macrophage+recruitment+and+polarization.">Human amnion epithelial cells mediate lung repair by directly modulating macrophage recruitment and polarization.</a> , (2013).</li><li>Litvinov, S. V., 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=Epithelial+cell+adhesion+molecule+(Ep-CAM)+modulates+cell-cell+interactions+mediated+by+classic+cadherins.">Epithelial cell adhesion molecule (Ep-CAM) modulates cell-cell interactions mediated by classic cadherins.</a> J Cell Biol. 139 (5), 1337-1348 (1997).</li><li>Winter, M. J., Nagtegaal, I. D., van Krieken, J. H., Litvinov, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epithelial+cell+adhesion+molecule+(Ep-CAM)+as+a+morphoregulatory+molecule+is+a+tool+in+surgical+pathology.">The epithelial cell adhesion molecule (Ep-CAM) as a morphoregulatory molecule is a tool in surgical pathology.</a> The American journal of pathology. 163 (6), 2139-2148 (2003).</li><li>Musina, R. A., Bekchanova, E. S., Sukhikh, G. T. <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+mesenchymal+stem+cells+obtained+from+different+human+tissues.">Comparison of mesenchymal stem cells obtained from different human tissues.</a> Bull Exp Biol Med. 139 (4), 504-509 (2005).</li><li>Park, 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=.">.</a> Newborn Stem Cells: Identity, Function, and Clinical Potential. , 119-137 (2013).</li></ol>

Bu metodolojinin başarısında önemli bir etkiye sahip birkaç kritik parametreler vardır. HAECs izolasyonundan önce 3 saat süre ile plasenta veya amniyon depolanması ancak doku mümkün olan en kısa zamanda işlem önerilmektedir, lojistik ve programlama amacıyla arzu edilebilir. Doku saklanacaksa, bu depolama amniyon membran diseksiyon ve yıkama sonrası yapılması tavsiye edilir. Amniyon sıvısı 4 ° C&#39;de steril HBSS içeren antibiyotik saklanabilir ancak hücre canlılığı, uzun depolama süresi il...

Plasenta gibi perinatal kaynaklardan, plasental membranlar, göbek kordonu ve amniyotik sıvıdan elde edilen hücreler rejeneratif tıp 1,2 için hücrelerin bir potansiyel kaynağı olarak araştırmacılar ve klinisyenler dikkatini çekmiştir. Bu ilginin nedeni, bu hücre tipleri, tüm plastisite ve bağışıklık yeteneği 3, potansiyel terapötik uygulamalar için temel olan bazı özellikleri derecesine sahip olmasıdır. hAECs rejeneratif hücre malzemeden bir miktarda potansiyel kaynağı temin terimi ya da önceden dönem amniyon zar 4 elde edilebilir heterojen epitel nüfus bulunmaktadır. hücresel bir tedavi olarak çekici hAECs yapmak özellikleri kendi multipotency, düşük imünojenite ve anti-inflamatuar özelliklerini içerir. hAECs (yüksek ölçüde, in vitro ve in vivo olarak multipotent mezodermal soy içine türetme yeteneğinde olduğu kardiyomiyopati bulunmuşturyocytes, myocytes, osteositler, adiposit), Endodermal soy (pankreas hücreleri, karaciğer hücreleri, akciğer hücreleri) ve ektodermal soyları (saç, cilt, sinir hücreleri ve astrositler) 5-10. Reassuringly kendi multipotency hAECs ya bir şekilde tümörlere görünür ya da in vivo olarak tümör gelişimini teşvik etmemesine karşın. Ayrıca, hAECs de sınıf II insan lökosit antijenleri (HLA&#39;lar) 8 düşük seviyelerde ifade, ayrıcalıklı bağışıklık bulunmaktadır. Bu özellik muhtemelen bağışıklık yetkili maymunlar, tavşan, kobay, sıçan, ve domuzlar 11-13 kullanıldığı çalışmalarda gösterildiği gibi, allojenik ve ksenojenik transplantasyon sonrası immün ret kaçınmak için kendi yeteneğini temelini oluşturmaktadır. hAECs güçlü immünomodülatör ve immünosüpresif özellikler gösterir ve böylece otoimmün hastalığın tedavisinde potansiyel klinik uygulamalar için önemli bir pratik avantaj sağlar. hAECs hem doğal ve adaptif bağışıklık sistemi üzerinde bağışıklık fonksiyonlarını uygulamak inanılmaktadır. Üzerindeimmunomodülator salgılanması 14 faktörleri ile öne mekanizmaların e. Klinik öncesi hayvan hastalık modellerinde hAECs Mevcut uygulamalar felç, multipl skleroz, karaciğer hastalığı, şeker hastalığı ve kronik ve akut akciğer hastalıkların tedavisi bulunmaktadır. Araştırmacılar nedeniyle benzersiz özellikleri inme sonrası beyin iltihabı tedavi etmek hAECs kullanarak ilgi gösterdi. HAECs, onlar aşılamak olabilir kan beyin bariyerini kadar 60 gün boyunca hayatta, nöronlar içine ayırt, inflamasyonu azaltmak ve nörolojik hastalıkların 15 hayvan modellerinde hasarlı santral sinir sistemi dokusunun yenilenmesini teşvik edebilir dair kanıtlar vardır. hAECs hedef ve multipl skleroz gelişmesi ve ilerlemesine katkıda birden patolojik yollar ters yeteneği sunuyoruz. Örneğin, klinik öncesi hayvan çalışmalarında kaynaklanan hAECs kuvvetle bağışıklık olduğunu göstermektedir vepotansiyel çevresel immün tolerans neden ve devam eden inflamatuar yanıtları tersine çevirebilirsiniz. hAECs in vivo sinir hücrelerine dönüştürmek ve nörotropik geniş bir dizi 16 faktörleri salgılayarak endojen sinir sistemini geliştirmek için kapasitesine sahip olduğu gösterilmiştir. İnsan ve kemirgen amniyon epitel hücreleri, hayvan modellerinde karaciğer hastalığının tedavisi için terapötik etkinliğini göstermiştir. Düşük hepatosit apoptoz eşliğinde karaciğer hastalığı, karaciğer canlı hAECs nakledilmesiyle için haec nakli kurşun, karbon tetraklorür hasar indüksiyon modelinde ve hepatik inflamasyon ve fibrozis 17 azalmıştır. hAECs insülin ve glükoz taşıyıcıları içeren ifade pankreas faktörlere stimüle edilebilir. Çeşitli çalışmalar, diyabetik farelerde 18 kan şekeri seviyelerinin normale dönmesi için hAECs potansiyelini araştırdık. Farelerde almahAECs, her iki hayvan vücut ağırlığı ve kan glukoz seviyeleri, hücre enjeksiyonunun ardından, normal seviyelere düşmüştür. Bu çalışmalar, diyabet tedavisi için hAECs kullanımı için güçlü bir sunulmaktadır. hAECs yetişkin ve yenidoğan modelleri 19 hem de önlenmesi ve deneysel akut ve kronik akciğer hasarı tamir kanıtlanmış rolü vardır. Bu çalışmalar, hAECs Kistik fibroz membran geçirgenliğinin düzenleyicisi (CFTR), kistik fibroz 20 hastalarda mutasyona uğramış, ve iyon kanalının de dahil olmak üzere birden fazla akciğer ilişkili proteinler ifade eden fonksiyonel akciğer epitel hücrelerine in vitro olarak ayırt bulundu. HAECs yaralı yetişkin ve yeni doğan akciğer teslim edilir, ayrıca, bunların nötrofil, makrofajlar ve lenfositler 21-23 dahil olmak üzere akciğer lökosit işe, azaltılması, konak bağışıklık hücrelerinin modülasyonu yoluyla onarım etkilerini uygularlar. Onların bolluğu göz önüne alındığında,Emniyet kaydı ve birden hastalıklar için kanıtlanmış klinik uygulamalar, hAECs kullanarak klinik çalışmalarda kaçınılmazdır. Klinik denemeler içine haec tedavilerin çeviri hızlandırılması amacı ile, biz mevcut iyi üretim uygulamaları (cGMP) kurallarına uygun olarak hayvansal ürün içermeyen reaktifler kullanılarak, klinik çalışmalar için uygun bir şekilde kültür hAECs, izole cryopreserve ve yöntemler geliştirdi . Biz bu protokolünü biz hayvansal kaynaklı reaktifler 6 kullanarak hAECs izole başarıyla kullandığınız bir önceden yayınlanmış bir protokol tabanlı. Biz hayvansal ürün içermeyen reaktif ile hayvansal kaynaklı ürünler yerine orijinal protokol değişmiş ve sonraki optimizasyon hücre verimi, canlılığı ve saflık optimize etmek yapıldı. Amacımız, insan klinik denemeler için hücre üretimi için düzenleyici standartlara uygun bir protokol geliştirmekti.

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			<td height="21" style="height:21px;">Soybean Trypsin Inhibitor&#160; 1g/50mL</td>
			<td style="width:119px;">Sigma Aldrich</td>
			<td style="width:119px;">T6522</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryostor CS5</td>
			<td style="width:119px;">BioLife Solutions</td>
			<td align="right" style="width:119px;">205102</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Trypan blue reagent</td>
			<td style="width:119px;">Life Technologies</td>
			<td style="width:119px;">15250-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-EpCam-PE</td>
			<td style="width:119px;">Miltenyi Biotec</td>
			<td style="width:119px;">130 - 091-253</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE-isotype control</td>
			<td style="width:119px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-098-845</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-CD90-PeCy5</td>
			<td style="width:119px;">BD Pharmingen</td>
			<td align="right" style="width:119px;">555597</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PeCy5-isotype control</td>
			<td style="width:119px;">BD Pharmingen</td>
			<td align="right" style="width:119px;">557224</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-CD105-APC</td>
			<td style="width:119px;">BD Pharmingen</td>
			<td align="right" style="width:119px;">562408</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">APC-isotype control</td>
			<td style="width:119px;">BD Pharmingen</td>
			<td align="right" style="width:119px;">340754</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagen Type VI</td>
			<td style="width:119px;">Sigma Aldrich</td>
			<td>C7521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Consumables</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50mL graduated pipette</td>
			<td style="width:119px;">BD/Falcon</td>
			<td>356550</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10mL graduated pipette</td>
			<td style="width:119px;">BD/Falcon</td>
			<td>356551</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5mL graduated pipette</td>
			<td style="width:119px;">BD/Falcon</td>
			<td>356543</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50mL falcon tubes</td>
			<td style="width:119px;">BD/Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15mL falcon tubes</td>
			<td style="width:119px;">BD/Falcon</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15-cm petri dishes</td>
			<td style="width:119px;">Corning</td>
			<td align="right" style="width:119px;">351058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70-&#956;m filters</td>
			<td style="width:119px;">BD/Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.22-&#956;m filters</td>
			<td style="width:119px;">Millipore</td>
			<td>SLGV033RS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">1ml Pipette tips</td>
			<td style="width:119px;">Fisherbrand</td>
			<td style="width:119px;">02-707-401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">200ul Pipette tips</td>
			<td style="width:119px;">Fisherbrand</td>
			<td style="width:119px;">02-707-409</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">20ml Syringe</td>
			<td style="width:119px;">BD/Medical</td>
			<td align="right" style="width:119px;">309661</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Plastic spatula</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>14-245-97&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Plastic weighing boat</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>02-202-102&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Cryo vials</td>
			<td>Nunc</td>
			<td>377267</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;"></td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;"></td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Equipment</td>
			<td style="width:119px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Mr Frosty</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>A451-4&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Biohazard Cabinet</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Bu prosedür doğru devam edildiğinde, 120,000,000 hAECs bir ortalama verim 80-160,000,000 hücre tipik aralık, beklenmelidir. Bu verimi kaynaktan, 83 ±% 4 arasında bir ortalama canlılığı beklenebilir. Klinik yöntemde artan ortalama verimi ve biraz daha düşük bir canlılık bağlı olarak serum proteinleri olmaması belki de, hayvan türevi ürün daha yüksek bir tripsin aktivitesi nedeniyle olabilir ve. İzole hAECs &lt;% 1, CD90, CD105, mezenkimal geni taşıyan hücreler ile% 92 EpCAM pozitif hücreleri ...

Watch this Scientific Journal Video about Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications at JoVE.com

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

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.

İzolasyon, Kriyoprezervasyon ve Kültür Klinik Uygulamaları İnsan Amniyon Epitel Hücreleri

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

Research

JoVE Journal

Medicine

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

İnsan Diş Pulp Kök Hücre İzolasyonu, Karakterizasyonu ve Karşılaştırmalı Farklılaşma İki Farklı Yöntemler Kullanılarak Daimi Dişler türetilen

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Tıp

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Hasta Türetilen Hücre Kültürü ve CD133 İzolasyonu<sup&gt; +</supMelanom dan&gt; sağlayan olası Kanser Kök Hücreleri

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

İnsan Fetal Pankreas Hücre Kümeleri İzolasyon, Kültür ve Görüntüleme

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Klinik ve Klinik Öncesi Araştırma Uygulamaları Tümör Hücre (CTC) Tahliller Sirkülasyon semiautomated adaptasyon

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Tümör spesifik Endotel Hücreleri İzolasyon ve Kültürü Genişleme

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

İnsan Hipofiz Adenom Hücreleri üç boyutlu Aljinat-boncuk Kültür

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Köpek arter ve venlerin Primer Endotel Hücreleri İzolasyon ve Kültür

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

A Novel Clinical Grade Isolation Method for Human Kidney Perivascular Stromal Cells

İnsan böbrek Perivasküler Stromal hücreler için bir roman klinik sınıf izolasyon yöntemi

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and ...

Culture and Imaging of Human Nasal Epithelial Organoids

İnsan Nazal Epitel Organoidlerinin Kültürü ve Görüntülenmesi

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis ...

Isolation and Culture of Human Adipose-Derived Stem Cells With an Innovative Xenogeneic-Free Method for Human Therapy

İnsan Tedavisi için Yenilikçi Bir Ksenojenik İçermeyen Yöntemle İnsan Adipoz Türevi Kök Hücrelerin İzolasyonu ve Kültürü

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission ...