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

We waarderen de steun van Mizuno Takako voor IFC- en H- en E-kleuring, Vanessa Garcia voor weefselsectie en Anika S Chandrasekaran voor hulp bij het voorbereiden van manuscripten. Dit werk wordt ondersteund door National Institutes of Health (5RO1HL135163-04, PO1HL108793-08) en Celgene IDEAL Consortium.

Menselijk longweefsel werd verkregen van overleden weefseldonoren in overeenstemming met toestemmingsprocedures ontwikkeld door het International Institute for the Advancement of Medicine (IIAM) en goedgekeurd door de Cedars-Sinai Medical Center Internal Review Board.
1. Weefselverwerking voor isolatie van longcellen uit tracheo-bronchiale of kleine luchtweg/parenchymale (kleine luchtwegen en longblaasjes) regio's
<ol><li>Bereid en autoclaaf alle dissectie-instrumenten, glaswerk en de juiste...

<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>

We beschrijven een betrouwbare methode voor de isolatie van gedefinieerde subpopulaties van longcellen uit menselijk longweefsel voor moleculaire of functionele analyse en ziektemodellering. Kritieke elementen van methoden zijn onder meer het vermogen om weefseldissociatie te bereiken met behoud van oppervlakte-epitopen, die antilichaam-gemedieerde verrijking van vers geïsoleerde cellen mogelijk maken, en de optimalisatie van kweekmethoden voor de efficiënte generatie van regiospecifieke epitheliale organoïden. We ric...

Luchtruimen van het menselijke ademhalingssysteem kunnen grofweg worden onderverdeeld in geleidende en respiratoire zones die respectievelijk het transport van gassen en hun daaropvolgende uitwisseling over de epitheliaal-microvasculaire barrière bemiddelen. De geleidende luchtwegen omvatten luchtpijpen, bronchiën, bronchiolen en terminale bronchiolen, terwijl ademhalingsluchtruimten respiratoire bronchiolen, alveolaire kanalen en longblaasjes omvatten. De epitheliale bekleding van deze luchtruimen verandert in samenstelling langs de proximo-distale as om tegemoet te komen aan de unieke vereisten van elke functioneel verschillende zone. Het pseudostratified epitheel van tracheo-bronchiale luchtwegen bestaat uit drie belangrijke celtypen, basaal, secretoir en trilhaar, naast de minder overvloedige celtypen waaronder borstel, neuro-endocriene en ionocyt 1,2,3. Bronchiolaire luchtwegen herbergen morfologisch vergelijkbare epitheelceltypen, hoewel er verschillen zijn in hun overvloed en functionele eigenschappen. Basale cellen zijn bijvoorbeeld minder overvloedig in bronchiolaire luchtwegen en secretoire cellen omvatten een groter aandeel clubcellen versus sereuze en bokaalcellen die overheersen in tracheo-bronchiale luchtwegen.  Epitheelcellen van de ademhalingszone omvatten een slecht gedefinieerd cuboidaal celtype in respiratoire bronchiolen, naast alveolaire type I (ATI) en type II (ATII) cellen van alveolaire kanalen en longblaasjes 1,4.
De identiteit van epitheelstam- en voorloperceltypen die bijdragen aan het behoud en de vernieuwing van epithelia in elke zone zijn onvolledig beschreven en grotendeels afgeleid uit studies in diermodellen 5,6,7,8. Studies bij muizen hebben aangetoond dat basale cellen van pseudostratified airways, of clubcellen van bronchiolaire luchtwegen of ATII-cellen van het alveolaire epitheel, dienen als epitheelstamcellen op basis van capaciteit voor onbeperkte zelfvernieuwing en multipotente differentiatie 7,9,10,11,12 . Ondanks het onvermogen om genetische afstammingstraceringsstudies uit te voeren om de stamheid van menselijke longepitheelceltypen te beoordelen, biedt de beschikbaarheid van op organoïden gebaseerde kweekmodellen om het functionele potentieel van epitheliale stam- en voorlopercellen te beoordelen een hulpmiddel voor vergelijkende studies tussen muis en mens 13,14,15,16,17.
We beschrijven methoden voor de isolatie van epitheelceltypen uit verschillende regio's van de menselijke long en hun cultuur met behulp van een 3D-organoïde systeem om de regionale celtypen samen te vatten. Soortgelijke methoden zijn ontwikkeld voor de functionele analyse en ziektemodellering van epitheelcellen uit andere orgaansystemen 18,19,20,21. Deze methoden bieden een platform voor de identificatie van regionale epitheliale voorlopercellen, om mechanistische studies uit te voeren die hun regulatie en micro-omgeving onderzoeken, en om ziektemodellering en medicijnontdekking mogelijk te maken. Hoewel studies van longepitheelvoorlopercellen uitgevoerd in diermodellen baat kunnen hebben bij de analyse, hetzij in vivo of in vitro, zijn inzichten in de identiteit van menselijke longepitheelvoorlopercellen grotendeels afhankelijk van extrapolatie van modelorganismen. Als zodanig bieden deze methoden een brug om de identiteit en het gedrag van menselijke longepitheelceltypen te relateren aan hun studies die de regulatie van stam- / voorlopercellen onderzoeken.

<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

Epitheliale organoïde modellen dienen als waardevolle hulpmiddelen om de basisbiologie van een orgaansysteem te bestuderen en voor ziektemodellering. Wanneer ze als organoïden worden gekweekt, kunnen epitheliale voorlopercellen zichzelf vernieuwen en onderscheidende nakomelingen genereren die cellulaire functies vertonen die vergelijkbaar zijn met die van hun in vivo tegenhangers. Hierin beschrijven we een stapsgewijs protocol om regiospecifieke voorlopercellen uit menselijke longen te isoleren en 3D-organoïde culturen te genereren als een experimenteel en validatie-instrument. We definiëren proximale en distale gebieden van de long met als doel regiospecifieke voorlopercellen te isoleren. We gebruikten een combinatie van enzymatische en mechanische dissociatie om totale cellen uit de long en luchtpijp te isoleren. Specifieke voorlopercellen werden vervolgens gefractioneerd uit de proximale of distale oorsprongscellen met behulp van fluorescentie geassocieerde celsortering (FACS) op basis van celtypespecifieke oppervlaktemarkers, zoals NGFR voor het sorteren van basale cellen en HTII-280 voor het sorteren van alveolaire type II-cellen. Geïsoleerde basale of alveolaire type II-voorlopers werden gebruikt om 3D-organoïde culturen te genereren. Zowel distale als proximale voorlopercellen vormden organoïden met een kolonievormende efficiëntie van 9-13% in distale regio en 7-10% in proximale regio bij verguld 5000 cel / put op dag 30. Distale organoïden handhaafden HTII-280+ alveolaire type II-cellen in cultuur, terwijl proximale organoïden op dag 30 differentieerden in trilhaar- en secretoire cellen. Deze 3D organoïde culturen kunnen worden gebruikt als een experimenteel hulpmiddel voor het bestuderen van de celbiologie van longepitheel en epitheliale mesenchymale interacties, evenals voor de ontwikkeling en validatie van therapeutische strategieën gericht op epitheliale disfunctie bij een ziekte.

Bron longweefsel De luchtpijp en extrapulmonale bronchus (figuur 1A) werden gebruikt als bronweefsel voor isolatie van proximale luchtwegepitheelcellen en de daaropvolgende generatie proximale organoïden. Distale longweefsels met zowel parenchym als kleine luchtwegen met een diameter van minder dan 2 mm (figuur 1A) werden gebruikt voor de isolatie van kleine luchtweg- en alveolaire epitheelcellen (distal longepitheel) en het genereren van kl...

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

Dit artikel biedt een gedetailleerde methodologie voor weefseldissociatie en cellulaire fractioneringsbenaderingen die verrijking van levensvatbare epitheelcellen uit proximale en distale gebieden van de menselijke long mogelijk maken. Hierin worden deze benaderingen toegepast voor de functionele analyse van longepitheelvoorlopercellen door het gebruik van 3D organoïdencultuurmodellen.

Isolatie en verrijking van menselijke longepitheelvoorlopercellen voor organoïde cultuur

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 

Isolatie en Cultuur van Post-Natal Mouse Cerebellaire Korrel Neuron progenitor cellen en Neuronen

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

Isolatie en grootschalige expansie van volwassen humane endotheelcellen kolonievormende Progenitor Cellen

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

Isolatie en Cultuur van Aviaire Embryonic Valvulaire Progenitor Cellen

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

Isolatie en Cultuur van Volwassen epitheliale stamcellen uit menselijke huid

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 Cultuur van primaire menselijke eileider epitheelcellen

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

Isolatie van Borst epitheelcellen van drie-dimensionale Mixed-cel Spheroid Co-cultuur

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

Seriële Verrijking van spermatogonia stam-en progenitorcellen (SSC&#39;s) in cultuur voor het afleiden van lange termijn Adult Mouse SSC Lines

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

Inductie en analyse van epitheliale naar mesenchymale transitie

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

Kweken van Menselijke neus epitheelcellen op de Air Liquid Interface

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

Isolatie en Cultuur van Dental epitheliale stamcellen van de volwassen muis Incisor

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