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

Apprezziamo il supporto di Mizuno Takako per la colorazione IFC e H ed E, Vanessa Garcia per il sezionamento dei tessuti e Anika S Chandrasekaran per l'aiuto nella preparazione del manoscritto. Questo lavoro è supportato dal National Institutes of Health (5RO1HL135163-04, PO1HL108793-08) e dal Celgene IDEAL Consortium.

Il tessuto polmonare umano è stato ottenuto da donatori di tessuto deceduti in conformità con le procedure di consenso sviluppate dall'International Institute for the Advancement of Medicine (IIAM) e approvate dal Cedars-Sinai Medical Center Internal Review Board.
1. Trattamento tissutale per l'isolamento di cellule polmonari da regioni tracheo-bronchiali o piccole vie aeree / parenchimali (piccole vie aeree e alveoli)
<ol><li>Preparare e autoclave tutti gli strumenti di dissezione, vetrer...

<ol>
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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. 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Descriviamo un metodo affidabile per l'isolamento di sottopopolazioni definite di cellule polmonari dal tessuto polmonare umano per l'analisi molecolare o funzionale e la modellazione della malattia. Gli elementi critici dei metodi includono la capacità di ottenere la dissociazione dei tessuti con la conservazione degli epitopi superficiali, che consentono l'arricchimento mediato da anticorpi di cellule appena isolate, e l'ottimizzazione dei metodi di coltura per la generazione efficiente di organoidi epiteliali specifi...

Gli spazi aerei del sistema respiratorio umano possono essere ampiamente suddivisi in zone conduttrici e respiratorie che mediano rispettivamente il trasporto di gas e il loro successivo scambio attraverso la barriera epiteliale-microvascolare. Le vie aeree conduttrici comprendono trachea, bronchi, bronchioli e bronchioli terminali, mentre gli spazi dell'aria respiratoria comprendono bronchioli respiratori, dotti alveolari e alveoli. Il rivestimento epiteliale di questi spazi aerei cambia nella composizione lungo l'asse prossima-distale per soddisfare i requisiti unici di ogni zona funzionalmente distinta. L'epitelio pseudostratificato delle vie aeree tracheo-bronchiali è composto da tre tipi di cellule principali, basale, secretoria e ciliata, oltre ai tipi cellulari meno abbondanti tra cui pennello, neuroendocrino e ionocita 1,2,3. Le vie aeree bronchiolari ospitano tipi di cellule epiteliali morfologicamente simili, sebbene vi siano distinzioni nella loro abbondanza e proprietà funzionali. Ad esempio, le cellule basali sono meno abbondanti all'interno delle vie aeree bronchiolari e le cellule secretorie includono una percentuale maggiore di cellule club rispetto alle cellule sierose e calici che predominano nelle vie aeree tracheo-bronchiali.  Le cellule epiteliali della zona respiratoria includono un tipo di cellule cuboidali scarsamente definito nei bronchioli respiratori, oltre alle cellule alveolari di tipo I (ATI) e di tipo II (ATII) dei dotti alveolari e degli alveoli 1,4.
L'identità dei tipi di cellule staminali epiteliali e progenitrici che contribuiscono al mantenimento e al rinnovamento degli epiteli in ciascuna zona è descritta in modo incompleto e ampiamente dedotta da studi su modelli animali 5,6,7,8. Studi sui topi hanno dimostrato che le cellule basali delle vie aeree pseudostratificate, o le cellule club delle vie aeree bronchiolari o le cellule ATII dell'epitelio alveolare, fungono da cellule staminali epiteliali basate sulla capacità di auto-rinnovamento illimitato e differenziazione multipotente 7,9,10,11,12 . Nonostante l'incapacità di eseguire studi di tracciamento del lignaggio genetico per valutare la staminalità dei tipi di cellule epiteliali polmonari umane, la disponibilità di modelli di coltura basati su organoidi per valutare il potenziale funzionale delle cellule staminali epiteliali e progenitrici fornisce uno strumento per studi comparativi tra topo e uomo 13,14,15,16,17.
Descriviamo metodi per l'isolamento di tipi di cellule epiteliali da diverse regioni del polmone umano e la loro coltura utilizzando un sistema organoide 3D per ricapitolare i tipi di cellule regionali. Metodi simili sono stati sviluppati per l'analisi funzionale e la modellazione della malattia di cellule epiteliali da altri sistemi di organi 18,19,20,21. Questi metodi forniscono una piattaforma per l'identificazione di cellule progenitrici epiteliali regionali, per eseguire studi meccanicistici che indagano la loro regolazione e microambiente e per consentire la modellazione della malattia e la scoperta di farmaci. Anche se gli studi sulle cellule progenitrici epiteliali polmonari eseguiti in modelli animali possono trarre beneficio dall'analisi, sia in vivo che in vitro, le intuizioni sull'identità delle cellule progenitrici epiteliali polmonari umane sono state in gran parte dipendenti dall'estrapolazione da organismi modello. Come tali, questi metodi forniscono un ponte per mettere in relazione l'identità e il comportamento dei tipi di cellule epiteliali polmonari umane con i loro studi che studiano la regolazione delle cellule staminali / progenitrici.

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			<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>
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		<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

I modelli organoidi epiteliali servono come strumenti preziosi per studiare la biologia di base di un sistema di organi e per la modellazione delle malattie. Se coltivate come organoidi, le cellule progenitrici epiteliali possono auto-rinnovarsi e generare progenie differenziante che mostra funzioni cellulari simili a quelle delle loro controparti in vivo . Qui descriviamo un protocollo passo-passo per isolare i progenitori specifici della regione dal polmone umano e generare colture organoidi 3D come strumento sperimentale e di convalida. Definiamo le regioni prossimali e distali del polmone con l'obiettivo di isolare le cellule progenitrici specifiche della regione. Abbiamo utilizzato una combinazione di dissociazione enzimatica e meccanica per isolare le cellule totali dal polmone e dalla trachea. Specifiche cellule progenitrici sono state quindi frazionate dalle cellule di origine prossimale o distale utilizzando lo smistamento cellulare associato alla fluorescenza (FACS) basato su marcatori di superficie specifici del tipo cellulare, come NGFR per lo smistamento delle cellule basali e HTII-280 per lo smistamento delle cellule alveolari di tipo II. I progenitori basali o alveolari isolati di tipo II sono stati utilizzati per generare colture organoidi 3D. Sia i progenitori distali che quelli prossimali formavano organoidi con un'efficienza di formazione della colonia del 9-13% nella regione distale e del 7-10% nella regione prossimale quando placcati 5000 cellule / pozzetto il giorno 30. Gli organoidi distali hanno mantenuto le cellule alveolari di tipo II HTII-280+ in coltura, mentre gli organoidi prossimali si sono differenziati in cellule ciliate e secretorie entro il giorno 30. Queste colture organoidiche 3D possono essere utilizzate come strumento sperimentale per lo studio della biologia cellulare dell'epitelio polmonare e delle interazioni mesenchimali epiteliali, nonché per lo sviluppo e la convalida di strategie terapeutiche mirate alla disfunzione epiteliale in una malattia.

Tessuto polmonare sorgente La trachea e il bronco extrapolmonare (Figura 1A) sono stati utilizzati come tessuto di origine per l'isolamento delle cellule epiteliali delle vie aeree prossimali e la successiva generazione di organoidi prossimali. Il tessuto polmonare distale che comprende sia il parenchima che le piccole vie aeree di diametro inferiore a 2 mm (Figura 1A) è stato utilizzato per l'isolamento di piccole cellule epiteliali delle v...

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

Questo articolo fornisce una metodologia dettagliata per la dissociazione dei tessuti e gli approcci di frazionamento cellulare che consentono l'arricchimento di cellule epiteliali vitali dalle regioni prossimali e distali del polmone umano. Qui questi approcci sono applicati per l'analisi funzionale di cellule progenitrici epiteliali polmonari attraverso l'uso di modelli di coltura di organoidi 3D.

Isolamento e arricchimento di cellule progenitrici epiteliali polmonari umane per la coltura di organoidi

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 

Isolamento e la coltura di cellule post-Natal Neuron cerebellare granuli mouse progenitore e Neuroni

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

Ricerca

Biologia

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

Isolamento e di espansione su vasta scala di umano adulto cellule progenitrici endoteliali che formano colonie

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

Isolamento e la coltura di cellule embrionali aviaria progenitrici valvolare

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

Isolamento e la coltura di cellule staminali epiteliali da pelle umana

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 Cultura Vivo di cellule primarie umane tube di Falloppio epiteliale

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

Isolamento di cellule epiteliali mammarie da tridimensionale Mixed-cell Spheroid Co-cultura

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

Arricchimento di serie staminali spermatogoni e cellule progenitrici (SSC) in Cultura per la derivazione di lungo termine Lines topo adulto SSC

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

Induzione e Analisi dei epiteliale a Mesenchymal Transizione

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

Coltura di cellule umane nasale epiteliali presso l&#39;Air Interface Liquid

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

Isolamento e Cultura della Dental epiteliali cellule staminali del topo adulto Incisor

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