Name: isolation and enrichment of human lung epithelial progenitor cells for organoid culture
Uploaded: 2020-07-21T14:00:00
Description: Watch this Scientific Journal Video about Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture at JoVE.com

IFC ve H ve E boyama için Mizuno Takako'nun, doku kesiti için Vanessa Garcia'nın ve el yazması hazırlanmasına yardımcı olduğu için Anika S Chandrasekaran'ın desteğine teşekkür ederiz. Bu çalışma Ulusal Sağlık Enstitüleri (5RO1HL135163-04, PO1HL108793-08) ve Celgene IDEAL Konsorsiyumu tarafından desteklenmektedir.

İnsan akciğer dokusu, ölen doku bağışçılarından, Uluslararası Tıbbın İlerlemesi Enstitüsü (IIAM) tarafından geliştirilen ve Cedars-Sinai Tıp Merkezi İç İnceleme Kurulu tarafından onaylanan rıza prosedürlerine uygun olarak elde edilmiştir.
1. Akciğer hücrelerinin trakeo-bronşiyal veya küçük hava yolu/parankimal (küçük hava yolları ve alveoller) bölgelerinden izolasyonu için doku işleme
<ol><li>Tüm diseksiyon aletlerini, cam eşyaları ve uygun çözeltil...

<ol>
	<li>Rackley, C. R., Stripp, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+and+maintaining+the+epithelium+of+the+lung.">Building and maintaining the epithelium of the lung.</a> Journal of Clinical Investigation. 122 (8), 2724-2730 (2012).</li><li>Montoro, D. 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=A+revised+airway+epithelial+hierarchy+includes+CFTR-expressing+ionocytes.">A revised airway epithelial hierarchy includes CFTR-expressing ionocytes.</a> Nature. 560 (7718), 319-324 (2018).</li><li>Plasschaert, L. W., 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+single-cell+atlas+of+the+airway+epithelium+reveals+the+CFTR-rich+pulmonary+ionocyte.">A single-cell atlas of the airway epithelium reveals the CFTR-rich pulmonary ionocyte.</a> Nature. 560 (7718), 377-381 (2018).</li><li>Barkauskas, C. E., 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=Type+2+alveolar+cells+are+stem+cells+in+adult+lung.">Type 2 alveolar cells are stem cells in adult lung.</a> Journal of Clinical Investigation. 123 (7), 3025-3036 (2013).</li><li>Barkauskas, C. E., 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=Lung+organoids:+current+uses+and+future+promise.">Lung organoids: current uses and future promise.</a> Development. 144 (6), 986-997 (2017).</li><li>Leeman, K. T., Fillmore, C. M., Kim, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+Stem+and+Progenitor+Cells+in+Tissue+Homeostasis+and+Disease.">Lung Stem and Progenitor Cells in Tissue Homeostasis and Disease.</a> Stem Cells in Development and Disease. 107, 207-233 (2014).</li><li>Rawlins, E. 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=The+Role+of+Scgb1a1(+)+Clara+Cells+in+the+Long-Term+Maintenance+and+Repair+of+Lung+Airway+but+Not+Alveolar,+Epithelium.">The Role of Scgb1a1(+) Clara Cells in the Long-Term Maintenance and Repair of Lung Airway but Not Alveolar, Epithelium.</a> Cell Stem Cell. 4 (6), 525-534 (2009).</li><li>Rock, J. 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=Basal+cells+as+stem+cells+of+the+mouse+trachea+and+human+airway+epithelium.">Basal cells as stem cells of the mouse trachea and human airway epithelium.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (31), 12771-12775 (2009).</li><li>Chang, W. 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=Bmp4+is+essential+for+the+formation+of+the+vestibular+apparatus+that+detects+angular+head+movements.">Bmp4 is essential for the formation of the vestibular apparatus that detects angular head movements.</a> Plos Genetics. 4 (4), 1000050 (2008).</li><li>McQualter, J. L., Bertoncello, I. <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:+Deconstructing+the+Lung+to+Reveal+Its+Regenerative+Potential.">Concise Review: Deconstructing the Lung to Reveal Its Regenerative Potential.</a> Stem Cells. 30 (5), 811-816 (2012).</li><li>Gonzalez, R. F., Allen, L., Gonzales, L., Ballard, P. L., Dobbs, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HTII-280,+a+Biomarker+Specific+to+the+Apical+Plasma+Membrane+of+Human+Lung+Alveolar+Type+II+Cells.">HTII-280, a Biomarker Specific to the Apical Plasma Membrane of Human Lung Alveolar Type II Cells.</a> Journal of Histochemistry &amp; Cytochemistry. 58 (10), 891-901 (2010).</li><li>Rock, J. 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=Notch-Dependent+Differentiation+of+Adult+Airway+Basal+Stem+Cells.">Notch-Dependent Differentiation of Adult Airway Basal Stem Cells.</a> Cell Stem Cell. 8 (6), 639-648 (2011).</li><li>Page, H., Flood, P., Reynaud, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+tissue+cultures:+current+trends+and+beyond.">Three-dimensional tissue cultures: current trends and beyond.</a> Cell and Tissue Research. 352 (1), 123-131 (2013).</li><li>Hynds, R. E., Giangreco, A. <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:+The+Relevance+of+Human+Stem+Cell-Derived+Organoid+Models+for+Epithelial+Translational+Medicine.">Concise Review: The Relevance of Human Stem Cell-Derived Organoid Models for Epithelial Translational Medicine.</a> Stem Cells. 31 (3), 417-422 (2013).</li><li>Lancaster, M. A., Knoblich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organogenesis+in+a+dish:+Modeling+development+and+disease+using+organoid+technologies.">Organogenesis in a dish: Modeling development and disease using organoid technologies.</a> Science. 345 (6194), (2014).</li><li>Weber, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoids+test+drug+response.">Organoids test drug response.</a> Nature Cell Biology. 20 (6), 634 (2018).</li><li>Fatehullah, A., Tan, S. H., Barker, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoids+as+an+in+vitro+model+of+human+development+and+disease.">Organoids as an in vitro model of human development and disease.</a> Nature Cell Biology. 18 (3), 246-254 (2016).</li><li>Nikolic, M. Z., Rawlins, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+Organoids+and+Their+Use+To+Study+Cell-Cell+Interaction.">Lung Organoids and Their Use To Study Cell-Cell Interaction.</a> Current Pathobiology Reports. 5 (2), 223-231 (2017).</li><li>Sato, 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=Long-term+expansion+of+epithelial+organoids+from+human+colon,+adenoma,+adenocarcinoma,+and+Barrett's+epithelium.">Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium.</a> Gastroenterology. 141 (5), 1762-1772 (2011).</li><li>Reynolds, B. A., Rietze, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cells+and+neurospheres--re-evaluating+the+relationship.">Neural stem cells and neurospheres--re-evaluating the relationship.</a> Nature Methods. 2 (5), 333-336 (2005).</li><li>Chua, C. W., 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=Single+luminal+epithelial+progenitors+can+generate+prostate+organoids+in+culture.">Single luminal epithelial progenitors can generate prostate organoids in culture.</a> Nature Cell Biology. 16 (10), 951-961 (2014).</li><li>Teisanu, R. 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=Functional+analysis+of+two+distinct+bronchiolar+progenitors+during+lung+injury+and+repair.">Functional analysis of two distinct bronchiolar progenitors during lung injury and repair.</a> American Journal of Respiratory and Cellular Molecular Biology. 44 (6), 794-803 (2011).</li><li>Chen, 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=Airway+epithelial+progenitors+are+region+specific+and+show+differential+responses+to+bleomycin-induced+lung+injury.">Airway epithelial progenitors are region specific and show differential responses to bleomycin-induced lung injury.</a> Stem Cells. 30 (9), 1948-1960 (2012).</li><li>Benam, K. 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=Small+airway-on-a-chip+enables+analysis+of+human+lung+inflammation+and+drug+responses+in+vitro.">Small airway-on-a-chip enables analysis of human lung inflammation and drug responses in vitro.</a> Nature Methods. 13 (2), 151-157 (2016).</li><li>Huh, 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=Reconstituting+organ-level+lung+functions+on+a+chip.">Reconstituting organ-level lung functions on a chip.</a> Science. 328 (5986), 1662-1668 (2010).</li><li>Jain, 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=Primary+Human+Lung+Alveolus-on-a-chip+Model+of+Intravascular+Thrombosis+for+Assessment+of+Therapeutics.">Primary Human Lung Alveolus-on-a-chip Model of Intravascular Thrombosis for Assessment of Therapeutics.</a> Clinical Pharmacology &amp; Therapeutics. 103 (2), 332-340 (2018).</li><li>Mulay, 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=SARS-CoV-2+infection+of+primary+human+lung+epithelium+for+COVID-19+modeling+and+drug+discovery.">SARS-CoV-2 infection of primary human lung epithelium for COVID-19 modeling and drug discovery.</a> bioRxiv. , (2020).</li></ol>

Moleküler veya fonksiyonel analiz ve hastalık modellemesi için insan akciğer dokusundan tanımlanmış akciğer hücrelerinin alt popülasyonlarının izolasyonu için güvenilir bir yöntem tanımladık. Yöntemlerin kritik unsurları, taze izole edilmiş hücrelerin antikor aracılı zenginleştirilmesine izin veren yüzey epitoplarının korunması ile doku ayrışmasını sağlama yeteneğini ve bölgeye özgü epitel organoidlerinin verimli bir şekilde üretilmesi için kültür yöntemlerinin optimizasyonunu i...

İnsan solunum sisteminin hava sahaları, sırasıyla gazların taşınmasına ve daha sonra epitel-mikrovasküler bariyer boyunca değişimine aracılık eden iletken ve solunum bölgelerine ayrılabilir. İletken hava yolları trakea, bronşlar, bronşiyoller ve terminal bronşiyolleri içerirken, solunum hava boşlukları solunum bronşiyolleri, alveoler kanallar ve alveolleri içerir. Bu hava sahalarının epitel astarı, fonksiyonel olarak farklı her bölgenin benzersiz gereksinimlerini karşılamak için proksimo-distal eksen boyunca kompozisyonda değişir. Trakeo-bronşiyal hava yollarının psödostratifiye epiteli, fırça, nöroendokrin ve iyonosit 1,2,3 dahil olmak üzere daha az miktarda hücre tipine ek olarak, bazal, salgı ve siliyer olmak üzere üç ana hücre tipinden oluşur. Bronşiyoler hava yolları, morfolojik olarak benzer epitel hücre tiplerini barındırır, ancak bolluklarında ve fonksiyonel özelliklerinde farklılıklar vardır. Örneğin, bazal hücreler bronşiyoler hava yollarında daha az bulunur ve salgı hücreleri, trakeo-bronşiyal hava yollarında baskın olan seröz ve kadeh hücrelerine karşı kulüp hücrelerinin daha büyük bir bölümünü içerir.  Solunum bölgesinin epitel hücreleri, alveoler kanalların ve alveollerin alveoler tip I (ATI) ve tip II (ATII) hücrelerine ek olarak, solunum bronşiyollerinde kötü tanımlanmış bir küboidal hücre tipini içerir 1,4.
Her bölgede epitelin korunmasına ve yenilenmesine katkıda bulunan epitel kök ve progenitör hücre tiplerinin kimliği eksik tanımlanmıştır ve büyük ölçüde hayvan modellerinde yapılan çalışmalardan çıkarılmıştır 5,6,7,8. Farelerde yapılan çalışmalar, psödotabakatize hava yollarının bazal hücrelerinin veya bronşiyoler hava yollarının kulüp hücrelerinin veya alveoler epitelin ATII hücrelerinin, sınırsız kendini yenileme ve multipotent farklılaşma kapasitesine dayanan epitel kök hücreleri olarak işlev gördüğünü göstermiştir 7,9,10,11,12 . İnsan akciğer epitel hücre tiplerinin köklülüğünü değerlendirmek için genetik soy izleme çalışmalarının yapılamamasına rağmen, epitel kök ve progenitör hücrelerin fonksiyonel potansiyelini değerlendirmek için organoid bazlı kültür modellerinin mevcudiyeti, fare ve insan arasındaki karşılaştırmalı çalışmalar için bir araç sağlamaktadır13,14,15,16,17.
Epitel hücre tiplerinin insan akciğerinin farklı bölgelerinden ve kültürlerinden izole edilmesi için bölgesel hücre tiplerini özetlemek için 3D organoid sistem kullanarak yöntemleri açıklıyoruz. Diğer organ sistemlerinden epitel hücrelerinin fonksiyonel analizi ve hastalık modellemesi için de benzer yöntemler geliştirilmiştir 18,19,20,21. Bu yöntemler, bölgesel epitel progenitör hücrelerinin tanımlanması, regülasyonlarını ve mikroçevrelerini araştıran mekanik çalışmalar yapmak, hastalık modellemesi ve ilaç keşfini sağlamak için bir platform sağlar. Hayvan modellerinde yapılan akciğer epitel progenitör hücreleri üzerine yapılan çalışmalar, in vivo veya in vitro analizden yararlanabilse de, insan akciğer epitelyal progenitör hücrelerinin kimliğine ilişkin bilgiler büyük ölçüde model organizmalardan ekstrapolasyona bağlı olmuştur. Bu nedenle, bu yöntemler, insan akciğer epitel hücre tiplerinin kimliğini ve davranışını, kök / progenitör hücrelerin düzenlenmesini araştıran çalışmaları ile ilişkilendirmek için bir köprü sağlar.

<table border="0" cellpadding="0" cellspacing="0" style="width:694px;" width="694">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">Cell Isolation</td>
			<td style="width:152px;"></td>
			<td style="width:161px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">10 mL Sterile syringes, Luer-Lok Tip</td>
			<td style="width:152px;">Fisher scientific</td>
			<td style="width:161px;">BD 309646</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">30 mL Sterile syringes, Luer-Lok Tip</td>
			<td style="width:152px;">VWR</td>
			<td style="width:161px;">BD302832</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">Biohazard bags</td>
			<td style="width:152px;">VWR</td>
			<td style="width:161px;">89495-440</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">Biohazard bags</td>
			<td style="width:152px;">VWR</td>
			<td style="width:161px;">89495-440</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">connecting ring</td>
			<td style="width:152px;">Pluriselect</td>
			<td style="width:161px;">41-50000-03</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">Deoxyribonuclease (lot#SLBF7798V)</td>
			<td style="width:152px;">sigma Aldrich</td>
			<td style="width:161px;">DN25-1G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">Disposable Petri dishes</td>
			<td style="width:152px;">Corning/Falcon</td>
			<td style="width:161px;">25373-187</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">Funnel</td>
			<td style="width:152px;">Pluriselect</td>
			<td style="width:161px;">42-50000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">HBSS</td>
			<td style="width:152px;">Corning</td>
			<td style="width:161px;">21-023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">Liberase TM Research Grade</td>
			<td style="width:152px;">sigma Aldrich</td>
			<td style="width:161px;">5401127001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">needle 16G</td>
			<td style="width:152px;">VWR</td>
			<td style="width:161px;">305198</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">needle 18G</td>
			<td style="width:152px;">VWR</td>
			<td style="width:161px;">305199</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:214px;">PluriStrainer 100 &#181;m (Cell Strainer)</td>
			<td style="width:152px;">Pluriselect</td>
			<td style="width:161px;">43-50100-51</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:214px;">PluriStrainer 300 &#181;m (Cell Strainer)</td>
			<td style="width:152px;">Pluriselect</td>
			<td style="width:161px;">43-50300-03</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:214px;">PluriStrainer 40 &#181;m (Cell Strainer)</td>
			<td style="width:152px;">Pluriselect</td>
			<td style="width:161px;">43-50040-51</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">PluriStrainer 500 &#181;m (Cell Strainer)</td>
			<td style="width:152px;">Pluriselect</td>
			<td style="width:161px;">43-50500-03</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:214px;">PluriStrainer 70 &#181;m (Cell Strainer)</td>
			<td style="width:152px;">Pluriselect</td>
			<td style="width:161px;">43-50070-51</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">Razor blades</td>
			<td style="width:152px;">VWR</td>
			<td style="width:161px;">55411-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">Red Blood Cell lysis buffer</td>
			<td style="width:152px;">eBioscience</td>
			<td style="width:161px;">00-4333-57</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">Equipment&#8217;s</td>
			<td style="width:152px;"></td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">GentleMACS C Tubes</td>
			<td style="width:152px;">MACS Miltenyi Biotec</td>
			<td style="width:161px;">130-096-334</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">GentleMACS Octo Dissociator</td>
			<td style="width:152px;">MACS Miltenyi Biotec</td>
			<td style="width:161px;">130-095-937</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">Leica ASP 300s Tissue processor</td>
			<td style="width:152px;"></td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">LS Columns</td>
			<td style="width:152px;">MACS Miltenyi Biotec</td>
			<td style="width:161px;">130-042-401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">MACS MultiStand**</td>
			<td style="width:152px;">Miltenyi Biotech</td>
			<td style="width:161px;">130-042-303</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">Thermomixer</td>
			<td style="width:152px;">Eppendorf</td>
			<td style="width:161px;">05-412-503</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:214px;">Thermomixer</td>
			<td style="width:152px;">Eppendorf</td>
			<td style="width:161px;">05-412-503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HBSS+ Buffer</td>
			<td style="width:152px;"></td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:214px;">Amphotericin B</td>
			<td style="width:152px;">Thermo fisher scientific</td>
			<td style="width:161px;">15290018</td>
			<td style="width:167px;">2ml</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">EDTA (0.5 M), pH 8.0, RNase-free</td>
			<td style="width:152px;">Thermo fisher scientific</td>
			<td style="width:161px;">AM9260G</td>
			<td style="width:167px;">500&#181;l</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Fetal Bovine Serum</td>
			<td style="width:152px;">Gemini Bio-Products</td>
			<td style="width:161px;">100-106</td>
			<td style="width:167px;">10ml</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">HBSS Hank&#39;s Balanced Salt Solution 1X 500 ml</td>
			<td style="width:152px;">VWR</td>
			<td style="width:161px;">45000-456</td>
			<td>500ml bottle</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">HEPES (1 M)</td>
			<td style="width:152px;">Thermo fisher scientific</td>
			<td style="width:161px;">15630080</td>
			<td style="width:167px;">5ml</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:214px;">Penicillin-Streptomycin-Neomycin (PSN) Antibiotic Mixture</td>
			<td style="width:152px;">Thermo fisher scientific</td>
			<td style="width:161px;">15640055</td>
			<td style="width:167px;">5ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">List of antibodies for FACS</td>
			<td style="width:152px;"></td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:214px;">Alexa Fluor 647 anti-human CD326 (EpCAM) Antibody</td>
			<td style="width:152px;">BioLegend</td>
			<td style="width:161px;">369820</td>
			<td style="width:167px;">1:50</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:214px;">BD CompBead Anti-Mouse Ig, K/ Negative control particles set</td>
			<td style="width:152px;">Fisher Scientific</td>
			<td style="width:161px;">BDB552843</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:214px;">CD31 MicroBead Kit, human</td>
			<td style="width:152px;">Miltenyi Biotec</td>
			<td style="width:161px;">130-091-935</td>
			<td style="width:167px;">20&#181;l/ 107 total cells</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:214px;">CD45 MicroBeads, human</td>
			<td style="width:152px;">Miltenyi Biotec</td>
			<td style="width:161px;">130-045-801</td>
			<td style="width:167px;">20&#181;l/ 107 total cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">DAPI</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:161px;">D9542-10MG</td>
			<td style="width:167px;">1:10000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">FITC anti-human CD235a</td>
			<td style="width:152px;">BioLegend</td>
			<td style="width:161px;">349104</td>
			<td style="width:167px;">1:100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">FITC anti-human CD31</td>
			<td style="width:152px;">BioLegend</td>
			<td style="width:161px;">303104</td>
			<td style="width:167px;">1:100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">FITC anti-human CD45</td>
			<td style="width:152px;">BioLegend</td>
			<td style="width:161px;">304054</td>
			<td style="width:167px;">1:100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">FITC anti-mouse IgM Antibody</td>
			<td style="width:152px;">BioLegend</td>
			<td style="width:161px;">406506</td>
			<td style="width:167px;">1:500</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">Mouse IgM anti human HT2-280</td>
			<td style="width:152px;">Terrace Biotech</td>
			<td style="width:161px;">TB-27AHT2-280</td>
			<td style="width:167px;">1:300</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">PE anti-human CD271(NGFR)</td>
			<td style="width:152px;">BioLegend</td>
			<td style="width:161px;">345106</td>
			<td style="width:167px;">1:50</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:367px;">Composition of Organoid Culture mediums</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">MRC-5</td>
			<td style="width:152px;">ATCC</td>
			<td style="width:161px;">CCL-171</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">PneumaCult -ALI Medium</td>
			<td style="width:152px;">Stemcell Technologies</td>
			<td style="width:161px;">5001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">Small Airway Epithelial Cell Growth Medium</td>
			<td style="width:152px;">PromoCell</td>
			<td style="width:161px;">C-21170</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">ThinCert Tissue Culture Inserts, Sterile</td>
			<td style="width:152px;">Greiner Bio-One</td>
			<td style="width:161px;">662641</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">Y-27632 (ROCK inhibitor) 100mM stock (1000x)</td>
			<td style="width:152px;">Stemcell Technologies</td>
			<td style="width:161px;">72302</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">Mouse Basal medium:</td>
			<td style="width:152px;"></td>
			<td style="width:161px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">Amphotericin B</td>
			<td style="width:152px;">Thermo fisher scientific</td>
			<td style="width:161px;">15290018</td>
			<td style="width:167px;">50 &#181;l</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">DMEM/F-12, HEPES</td>
			<td style="width:152px;">ThermoFisher scientific</td>
			<td style="width:161px;">11330032</td>
			<td style="width:167px;">50 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">Fetal Bovine Serum</td>
			<td style="width:152px;">Gemini Bio-Products</td>
			<td style="width:161px;">100-106</td>
			<td style="width:167px;">5 ml</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">Insulin-Transferrin-Selenium (ITS -G) (100X)</td>
			<td style="width:152px;">ThermoFisher scientific</td>
			<td style="width:161px;">41400045</td>
			<td style="width:167px;">500 &#181;l</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:214px;">Penicillin-Streptomycin-Neomycin (PSN) Antibiotic Mixture</td>
			<td style="width:152px;">Thermo fisher scientific</td>
			<td style="width:161px;">15640055</td>
			<td style="width:167px;">500 &#181;l</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">SB431542 TGF-&#946; pathway inhibitor (stock 100 mM)</td>
			<td style="width:152px;">Stem cell</td>
			<td style="width:161px;">72234</td>
			<td style="width:167px;">5 &#181;l</td>
		</tr>
		<tr height="42">
			<td colspan="2" height="42" style="height:42px;width:367px;">List of antibodies for Immunohistochemistry</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">Antigen unmasking solution, citric acid based</td>
			<td style="width:152px;">Vector</td>
			<td style="width:161px;">H-3300</td>
			<td style="width:167px;">937 &#181;l in 100ml water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">Histogel</td>
			<td style="width:152px;">Thermo Scientific</td>
			<td style="width:161px;">HG-4000-012</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">Primary Antibodies</td>
			<td style="width:152px;"></td>
			<td style="width:161px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">Anti HT2-280</td>
			<td style="width:152px;">Terracebiotech</td>
			<td style="width:161px;">TB-27AHT2-280</td>
			<td style="width:167px;">1:500</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">FOXJ1 Monoclonal Antibody (2A5)</td>
			<td style="width:152px;">Thermo Fisher Scientific</td>
			<td style="width:161px;">14-9965-82</td>
			<td style="width:167px;">1:300</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">Human Uteroglobin/SCGB1A1 Antibody</td>
			<td style="width:152px;">R and D systems</td>
			<td style="width:161px;">MAB4218</td>
			<td style="width:167px;">1:300</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">Keratin 5 Polyclonal Chicken Antibody, Purified [Poly9059]</td>
			<td style="width:152px;">Biolegend</td>
			<td style="width:161px;">905901</td>
			<td style="width:167px;">1:500</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">MUC5AC Monoclonal Antibody (45M1)</td>
			<td style="width:152px;">Thermo Fisher Scientific</td>
			<td style="width:161px;">MA5-12178</td>
			<td style="width:167px;">1:300</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">PDPN / Podoplanin Antibody (clone 8.1.1)</td>
			<td style="width:152px;">LifeSpan Biosciences</td>
			<td style="width:161px;">LS-C143022-100</td>
			<td style="width:167px;">1:300</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">Purified Mouse Anti-E-Cadherin</td>
			<td style="width:152px;">BD biosciences</td>
			<td style="width:161px;">610182</td>
			<td style="width:167px;">1:1000</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">Sox-2 Antibody</td>
			<td style="width:152px;">Santa Cruz biotechnologies</td>
			<td style="width:161px;">sc-365964</td>
			<td style="width:167px;">1:300</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">Secondary Antibodies</td>
			<td style="width:152px;"></td>
			<td style="width:161px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">Donkey anti-rabbit lgG, 488</td>
			<td style="width:152px;">Thermo Fisher Scientific</td>
			<td style="width:161px;">A-21206</td>
			<td style="width:167px;">1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">FITC anti-mouse IgM Antibody</td>
			<td style="width:152px;">BioLegend</td>
			<td style="width:161px;">406506</td>
			<td style="width:167px;">1:500</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">Goat anti-Hamster IgG (H+L), Alexa Fluor 594</td>
			<td style="width:152px;">Thermo Fisher Scientific</td>
			<td style="width:161px;">A-21113</td>
			<td style="width:167px;">1:500</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:214px;">Goat anti-Mouse IgG1 Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td style="width:152px;">Thermo Fisher Scientific</td>
			<td style="width:161px;">A-21121</td>
			<td style="width:167px;">1:500</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:214px;">Goat anti-Mouse IgG2a Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td style="width:152px;">Thermo Fisher Scientific</td>
			<td style="width:161px;">A-21131</td>
			<td style="width:167px;">1:500</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:214px;">Goat anti-Mouse IgG2a Cross-Adsorbed Secondary Antibody, Alexa Fluor 568</td>
			<td style="width:152px;">Thermo Fisher Scientific</td>
			<td style="width:161px;">A-21134</td>
			<td style="width:167px;">1:500</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:214px;">Goat anti-Mouse IgG2b Cross-Adsorbed Secondary Antibody, Alexa Fluor 568</td>
			<td style="width:152px;">Thermo Fisher Scientific</td>
			<td style="width:161px;">A-21144</td>
			<td style="width:167px;">1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">Buffers</td>
			<td style="width:152px;"></td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:214px;">Immunohistochemistry Blocking Solution</td>
			<td style="width:152px;"></td>
			<td style="width:161px;"></td>
			<td style="width:167px;">3% BSA, o.4% Triton-x100 in TBS (Tris based saline)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">Immunohistochemistry Incubation Solution</td>
			<td style="width:152px;"></td>
			<td style="width:161px;"></td>
			<td style="width:167px;">3% BSA, ).1% Triton-X100 in TBS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">Immunohistochemistry Washing Solution</td>
			<td style="width:152px;"></td>
			<td style="width:161px;"></td>
			<td style="width:167px;">TBS with 0.1% Tween 20</td>
		</tr>
	</tbody>
</table>

isolation and enrichment of human lung epithelial progenitor cells for organoid culture

Epitelyal organoid modelleri, bir organ sisteminin temel biyolojisini incelemek ve hastalık modellemesi için değerli araçlar olarak hizmet eder. Organoidler olarak yetiştirildiğinde, epitelyal progenitör hücreler kendi kendini yenileyebilir ve in vivo meslektaşlarınınkine benzer hücresel fonksiyonlar sergileyen farklılaştırıcı döller üretebilir. Burada, bölgeye özgü progenitörleri insan akciğerinden izole etmek ve deneysel ve doğrulama aracı olarak 3D organoid kültürler üretmek için adım adım bir protokol açıklanmaktadır. Akciğerin proksimal ve distal bölgelerini, bölgeye özgü progenitör hücreleri izole etmek amacıyla tanımlıyoruz. Toplam hücreleri akciğer ve trakeadan izole etmek için enzimatik ve mekanik ayrışmanın bir kombinasyonunu kullandık. Spesifik progenitör hücreler daha sonra, bazal hücreleri sıralamak için NGFR ve alveolar tip II hücreleri sıralamak için HTII-280 gibi hücre tipine özgü yüzey belirteçlerine dayanan floresan ilişkili hücre sıralama (FACS) kullanılarak proksimal veya distal orijinli hücrelerden fraksiyone edildi. 3D organoid kültürleri oluşturmak için izole bazal veya alveoler tip II progenitörler kullanıldı. Hem distal hem de proksimal progenitörler, 30. günde 5000 hücre/kuyu kaplandıklarında distal bölgede %9-13, proksimal bölgede %7-10 kolon oluşturan etkinliğe sahip organoidler oluşturdular. Distal organoidler kültürde HTII-280+ alveoler tip II hücrelerini korurken, proksimal organoidler 30. günde siliyer ve sekretuar hücrelere farklılaştı. Bu 3D organoid kültürler, akciğer epiteli ve epitel mezenkimal etkileşimlerinin hücre biyolojisini incelemek ve ayrıca bir hastalıkta epitel disfonksiyonunu hedefleyen terapötik stratejilerin geliştirilmesi ve doğrulanması için deneysel bir araç olarak kullanılabilir.

Kaynak akciğer dokusu Trakea ve ekstrapulmoner bronş (Şekil 1A) proksimal hava yolu epitel hücrelerinin ve sonraki jenerasyon proksimal organoidlerin izolasyonunda kaynak doku olarak kullanıldı. Hem parankimi hem de çapı 2 mm'den küçük küçük hava yollarını içeren distal akciğer dokusu (Şekil 1A), küçük hava yolu ve alveoler epitel hücrelerinin (distal akciğer epiteli) izolasyonu ve küçük hava yolu veya alveoler orga...

Watch this Scientific Journal Video about Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture at JoVE.com

Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture

Bu makalede, insan akciğerinin proksimal ve distal bölgelerinden canlı epitel hücrelerinin zenginleştirilmesine izin veren doku dissosiyasyonu ve hücresel fraksiyonasyon yaklaşımları için ayrıntılı bir metodoloji sunulmaktadır. Bu yaklaşımlar, akciğer epitel progenitör hücrelerinin fonksiyonel analizi için 3D organoid kültür modelleri kullanılarak uygulanmaktadır.

Organoid Kültür için İnsan Akciğer Epitel Progenitör Hücrelerinin İzolasyonu ve Zenginleştirilmesi

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, ...

Research

JoVE Journal

Biology

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons 

Post-Natal Mouse Serebellar Granül Nöron Progenitör Hücreler ve Nöronlar İzolasyon ve Kültür

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the ...

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

İzolasyon ve Büyük Ölçekli Genişleme Yetişkin İnsan Endotel Koloni Oluşturan Progenitör Hücreler

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Kuş Embriyonik Kapak Progenitör Hücreler izolasyonu ve Kültür

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

İnsan Cilt Yetişkin Epitel Kök Hücre İzolasyonu ve Kültür

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Ex Vivo Kültür İlköğretim İnsan Fallop Tüp Epitel Hücreleri

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

Üç boyutlu Karışık-hücre Sfero Co-kültürden Meme Epitel Hücrelerinin İzolasyonu

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

Uzun vadeli Yetişkin Fare SSC Hatları türetilmesi için Kültür Spermatogonial Kök ve progenitör hücrelerin Seri Zenginleştirme (DGM)

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Mezenkimal Geçiş indüksiyon ve Epitel Analizi

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

Hava Sıvı Arayüz İnsan Burun Epitel Hücrelerinin ekimi

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Yetişkin Fare insizör Diş Epitel Kök Hücreleri İzolasyon ve Kültür

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...