Agradecemos el apoyo de Mizuno Takako para IFC y tinción H y E, Vanessa García para la seccionamiento de tejidos y Anika S Chandrasekaran por ayudar con la preparación del manuscrito. Este trabajo cuenta con el apoyo de los Institutos Nacionales de Salud (5RO1HL135163-04, PO1HL108793-08) y el Consorcio Celgene IDEAL.

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Los autores no tienen nada que revelar.

Describimos un método confiable para el aislamiento de subpoblaciones definidas de células pulmonares del tejido pulmonar humano para análisis molecular o funcional y modelado de enfermedades. Los elementos críticos de los métodos incluyen la capacidad de lograr la disociación tisular con la preservación de epítopos superficiales, que permiten el enriquecimiento mediado por anticuerpos de células recién aisladas, y la optimización de los métodos de cultivo para la generación eficiente de organoides epitelial...

Los espacios aéreos del sistema respiratorio humano se pueden dividir ampliamente en zonas conductoras y respiratorias que median el transporte de gases y su posterior intercambio a través de la barrera epitelial-microvascular, respectivamente. Las vías respiratorias conductoras incluyen tráquea, bronquios, bronquiolos y bronquiolos terminales, mientras que los espacios de aire respiratorio incluyen bronquiolos respiratorios, conductos alveolares y alvéolos. El revestimiento epitelial de estos espacios aéreos cambia de composición a lo largo del eje proximo-distal para adaptarse a los requisitos únicos de cada zona funcionalmente distinta. El epitelio pseudoestratificado de las vías respiratorias traqueobronquiales está compuesto por tres tipos principales de células, basales, secretoras y ciliadas, además de los tipos celulares menos abundantes, incluidos el cepillo, el neuroendocrino y el ionocito 1,2,3. Las vías respiratorias bronquiolares albergan tipos de células epiteliales morfológicamente similares, aunque hay distinciones en su abundancia y propiedades funcionales. Por ejemplo, las células basales son menos abundantes dentro de las vías respiratorias bronquiolares, y las células secretoras incluyen una mayor proporción de células club frente a las células serosas y caliciformes que predominan en las vías respiratorias traqueobronquiales.  Las células epiteliales de la zona respiratoria incluyen un tipo de célula cuboidal mal definida en los bronquiolos respiratorios, además de las células alveolares tipo I (ATI) y tipo II (ATII) de los conductos alveolares y alvéolos 1,4.
La identidad de los tipos de células madre epiteliales y progenitoras que contribuyen al mantenimiento y renovación de los epitelios en cada zona se describe de forma incompleta y se infiere en gran medida de los estudios en modelos animales 5,6,7,8. Los estudios en ratones han demostrado que las células basales de las vías respiratorias pseudoestratificadas, o las células club de las vías respiratorias bronquiolares o las células ATII del epitelio alveolar, sirven como células madre epiteliales basadas en la capacidad de autorrenovación ilimitada y diferenciación multipotente 7,9,10,11,12 . A pesar de la incapacidad de realizar estudios de rastreo de linaje genético para evaluar el tallo de los tipos de células epiteliales pulmonares humanas, la disponibilidad de modelos de cultivo basados en organoides para evaluar el potencial funcional de las células madre y progenitoras epiteliales proporciona una herramienta para estudios comparativos entre ratones y humanos 13,14,15,16,17.
Describimos métodos para el aislamiento de tipos de células epiteliales de diferentes regiones del pulmón humano y su cultivo utilizando un sistema organoide 3D para recapitular los tipos de células regionales. Se han desarrollado métodos similares para el análisis funcional y el modelado de enfermedades de células epiteliales de otros sistemas de órganos 18,19,20,21. Estos métodos proporcionan una plataforma para la identificación de células progenitoras epiteliales regionales, para realizar estudios mecanicistas que investiguen su regulación y microambiente, y para permitir el modelado de enfermedades y el descubrimiento de fármacos. Aunque los estudios de células progenitoras epiteliales pulmonares realizados en modelos animales pueden beneficiarse del análisis, ya sea in vivo o in vitro, los conocimientos sobre la identidad de las células progenitoras epiteliales pulmonares humanas han dependido en gran medida de la extrapolación de organismos modelo. Como tal, estos métodos proporcionan un puente para relacionar la identidad y el comportamiento de los tipos de células epiteliales pulmonares humanas con sus estudios que investigan la regulación de las células madre / progenitoras.

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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>
		</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>
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		<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

Los modelos organoides epiteliales sirven como herramientas valiosas para estudiar la biología básica de un sistema de órganos y para el modelado de enfermedades. Cuando se cultivan como organoides, las células progenitoras epiteliales pueden autorrenovarse y generar una progenie diferenciadora que exhibe funciones celulares similares a las de sus contrapartes in vivo . A continuación describimos un protocolo paso a paso para aislar progenitores específicos de la región del pulmón humano y generar cultivos organoides 3D como herramienta experimental y de validación. Definimos las regiones proximales y distales del pulmón con el objetivo de aislar las células progenitoras específicas de la región. Utilizamos una combinación de disociación enzimática y mecánica para aislar las células totales del pulmón y la tráquea. Las células progenitoras específicas se fraccionaron de las células de origen proximal o distal utilizando la clasificación celular asociada a la fluorescencia (FACS) basada en marcadores de superficie específicos del tipo celular, como NGFR para clasificar las células basales y HTII-280 para clasificar las células alveolares tipo II. Se utilizaron progenitores basales o alveolares aislados de tipo II para generar cultivos organoides 3D. Tanto los progenitores distales como los proximales formaron organoides con una eficiencia de formación de colonias del 9-13% en la región distal y del 7-10% en la región proximal cuando se chapan 5000 células / pozo en el día 30. Los organoides distales mantuvieron las células alveolares tipo II HTII-280+ en cultivo, mientras que los organoides proximales se diferenciaron en células ciliadas y secretoras para el día 30. Estos cultivos organoides 3D se pueden utilizar como una herramienta experimental para estudiar la biología celular del epitelio pulmonar y las interacciones mesenquimales epiteliales, así como para el desarrollo y validación de estrategias terapéuticas dirigidas a la disfunción epitelial en una enfermedad.

Fuente de tejido pulmonar La tráquea y el bronquio extrapulmonar (Figura 1A) se utilizaron como tejido fuente para el aislamiento de las células epiteliales de las vías respiratorias proximales y la posterior generación de organoides proximales. Se utilizó tejido pulmonar distal que incluye tanto parénquima como vías respiratorias pequeñas de menos de 2 mm de diámetro (Figura 1A) para el aislamiento de células epiteliales alveolares...

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

Este artículo proporciona una metodología detallada para los enfoques de disociación tisular y fraccionamiento celular que permiten el enriquecimiento de células epiteliales viables de las regiones proximal y distal del pulmón humano. En este caso, estos enfoques se aplican para el análisis funcional de células progenitoras epiteliales pulmonares mediante el uso de modelos de cultivo de organoides 3D.

Aislamiento y enriquecimiento de células progenitoras epiteliales pulmonares humanas para cultivo de organoides

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

isolation-enrichment-human-lung-epithelial-progenitor-cells-for

Research

JoVE Journal

Biology

7.9K vistas.  Cedars-Sinai Medical Center. Este artículo proporciona una metodología detallada para los enfoques de disociación tisular y fraccionamiento celular que permiten el enriquecimiento de células epiteliales viables de las regiones proximal y distal del pulmón humano. En este caso, estos enfoques se aplican para el análisis funcional de células progenitoras epiteliales pulmonares mediante el uso de modelos de cultivo de organoides 3D.

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

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

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the cerebellar neuronal types (granule cells, Purkinje cells and inhibitory interneurons), granule neurons are by far the most numerous and are the most abundant type of neurons in the mammalian brain. In rodents, cerebellar granule neurons are generated during the first two post-natal weeks from progenitor cells in the outermost layer of the cerebellar cortex, the external granule layer (EGL). The protocol presented here describes techniques to enrich and culture granule neurons and their progenitor cells from post-natal mouse cerebellum. We will describe procedures to obtain cultures of increasing purity3,4, which can be used to study the differentiation of proliferating progenitor cells into granule neurons5,6. Once the progenitor cells differentiate, the cultures also provide a homogenous population of granule neurons for experimental manipulation and characterization of phenomena such as synaptogenesis, glutamate receptor function7, interaction with other purified cerebellar cells8,9 or cell death7.

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

Here we present a method to isolate and culture cerebellar granule neuron progenitor cells and cerebellar granule neurons from postnatal mouse.

Aislamiento y cultivo de Post-Natal células de ratón cerebelosa gránulo Progenitor neuronas y las neuronas

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

Investigación

Biología

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

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

El aislamiento y la expansión a gran escala de células humanas adultas Colonia progenitoras endoteliales formación

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

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

Aislamiento y cultivo de células embrionarias progenitoras aviar valvular

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

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

Aislamiento y cultivo de células madre adultas epiteliales de la piel humana

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

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 uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

Ex Vivo Cultura de células humanas primarias epiteliales del tubo de Falopio

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

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

Aislamiento de células epiteliales mamarias de tridimensional mixto de células Esferoide 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 ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

Aislamiento de la cromatina mediante la purificación de ARN (chirp)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term 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 the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells1-3.
Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines4. However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state.
Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors5,6. Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies7. Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types8. Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC2,3.
The method described below entails a simple procedure for deriving adult SSC lines by dissociating adult donor seminiferous tubules, followed by plating of cells on feeders comprised of a testicular stromal cell line (JK1)3. Through serial passaging, strongly adherent, contaminating non-germ cells are depleted from the culture with concomitant enrichment of SSCs. Cultures produced in this manner contain a mixture of spermatogonia at different stages of differentiation, which contain SSCs, based on long-term self renewal capability. The crux of the SESC method is that it enables SSCs to make the difficult transition from self-renewal in vivo to long-term self-renewal in vitro in a radically different microenvironment, produces long-term SSC lines, free of contaminating somatic cells, and thereby enables subsequent experimental manipulation of SSCs.

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

A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.

Enriquecimiento de serie de la madre de espermatogonias y células progenitoras (SSC) en Cultura para la derivación de largo plazo de ratones adultos Líneas 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

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Inducción y Análisis de epitelial a mesenquimal de transición

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

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

El cultivo de células humanas epiteliales nasales en la interfase aire líquido

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