Name: Författare i fokus: Utveckling och karakterisering av en in vitro-modell för att studera kronisk exponering för cigarettrök och dess inverkan på luftvägsepitelceller i KOL-forskning
Uploaded: 2024-07-12T14:50:00
Description: Chronic obstructive pulmonary disease (COPD) is largely attributed to tobacco smoke exposure. Investigating how airway epithelial cells functionally adapt ...

Denna studie stöddes av National Natural Science Foundation of China (82090013).

Det övergripande protokollet kräver 44 dagar, inklusive 1 dag för beredning av luftvägsepitelceller isolerade från murina luftstrupar, 15 dagar för cellproliferation och 28 dagar för CSE-stimulering vid luft-vätskegränssnittet. Alla försöksdjur är inrymda i SPF Barrier Animal Room på Animal Experiment Center of Capital Medical University och har granskats och godkänts av Animal Experiment and Laboratory Animal Ethics Committee of Capital Medical University (AEEI-2020-100) för att uppfylla kraven i ARRIVE-r...

In vitro-modell av murina luftvägsepitelceller och kronisk rökexponering

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+airway+epithelial+injury+and+abnormal+repair+in+asthma+and+COPD.">Mechanisms of airway epithelial injury and abnormal repair in asthma and COPD.</a> Front Immunol. 14, 1201658 (2023).</li><li>Zhou, J. -. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cigarette+smoke-initiated+autoimmunity+facilitates+sensitisation+to+elastin-induced+COPD-like+pathologies+in+mice.">Cigarette smoke-initiated autoimmunity facilitates sensitisation to elastin-induced COPD-like pathologies in mice.</a> Eur Respir J. 56 (3), 2000404 (2020).</li><li>Lam, H. C., Choi, A. M. K., Ryter, S. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucinlike+glycoproteins+from+cat+tracheal+gland+cells+in+primary+culture.">Mucinlike glycoproteins from cat tracheal gland cells in primary culture.</a> Am J Physiol. 265 (3 Pt 1), L260-L269 (1993).</li><li>Cao, X., 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=Invited+review:+human+air-liquid-interface+organotypic+airway+tissue+models+derived+from+primary+tracheobronchial+epithelial+cells-overview+and+perspectives.">Invited review: human air-liquid-interface organotypic airway tissue models derived from primary tracheobronchial epithelial cells-overview and perspectives.</a> In Vitro Cell Dev Biol Anim. 57 (2), 104-132 (2021).</li><li>Beb&#246;k, Z., Tousson, A., Schwiebert, L. M., Venglarik, C. 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KOL är en vanlig kronisk luftvägsinflammation. Exponering för tobaksrök leder till kronisk luftvägsinflammation, luftvägsombyggnad och strukturell förstörelse av lungorna, vilket är resultatet av interaktionen mellan olika strukturella celler och immunceller10. Som frontlinjen i det medfödda immunsystemet i lungan spelar luftvägsepitelceller en mycket viktig roll under utvecklingen av sjukdomen11. Ur denna synvinkel är det ett vetenskapligt problem som måste l?...

Kroniskt obstruktiv lungsjukdom (KOL) är en heterogen lungsjukdom med komplexa egenskaper, medan patienter med KOL gradvis tenderar att bliyngre1. Rökning, som är en primär riskfaktor för KOL2, har en djupgående inverkan på luftvägarnas epitelceller, som fungerar som den första barriären mot tobaksrök. Trots detta kända samband är de detaljerade mekanismerna genom vilka tobaksrök inducerar förändringar i luftvägarnas epitelceller fortfarande otillräckligt utforskade. En grundlig förståelse av dessa molekylära förändringar är avgörande för att identifiera tidiga diagnostiska markörer och terapeutiska mål för KOL.
För att ta itu med denna lucka utvecklade vi en ny in vitro-modell med hjälp av murina luftvägsepitelceller. Dessa celler utsattes för långvarig stimulering med cigarettrökextrakt (CSE), vilket gjorde det möjligt för oss att övervaka dynamiska cellulära förändringar och utforska förändringarna i luftvägsepitelcellerna under långvarig tobaksstimulering och den underliggande mekanismen. I denna modell användes transwell för att tillhandahålla luft-vätskegränssnittet för luftvägsepitelceller, och cigarettrökextrakt stimulerades i det tidiga stadiet av epitelcellsdifferentiering fram till slutet av differentieringen efter 28 dagar. Tidigare studier har endast undersökt differentiering och kortvarig stimulering av luftvägsepitelceller (cellinjedominans). De var begränsade till en enda regleringsväg 3,4,5,6. Protokollet som presenteras här använder dock primära luftvägsepitelceller från möss och optimerar cellextraktionsprocessen för bättre cellaktivitet än tidigare metoder för odling av luftvägsepitelceller. Denna modell fokuserar på förändringar i cellulär differentiering tillsammans med omfattande transkriptomiska, proteomiska, metabolomiska och epigenomiska analyser. Genom att använda immunofluorescens och avancerade multi-omics-tekniker syftade vi till att belysa de cellulära svaren hos luftvägsepitelceller på kronisk exponering för tobaksrök, och därigenom bidra till en djupare förståelse av KOL-patogenesen. Denna modell kan användas för att utforska förändringarna i luftvägsepitelcelldifferentieringsmönster och dess mekanism som orsakas av långvarig stimulering av olika föroreningar.

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	<tbody>
		<tr>
			<td>100x Penicillin/Streptomycin solution</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>24 mm Transwell with 0.4 &#181;m Pore Polyester Membrane Insert, Sterile</td>
			<td>BIOFIL</td>
			<td>TCS016012</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#181;m Cell Strainer</td>
			<td>Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>500x Gentamicin/Amphotericin Solution</td>
			<td>Gibco</td>
			<td>R01510</td>
			<td></td>
		</tr>
		<tr>
			<td>acetylated &#945;-Tubulin</td>
			<td>CST</td>
			<td>#5335</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetyl-&#945;-Tubulin (Lys40) (D20G3)XP Rabbit mAb&#160;</td>
			<td>cellsignal</td>
			<td>#5335</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal Component Free Cell Dissociation Kit</td>
			<td>Stemcell</td>
			<td>05426</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-pan Cytokeratin antibody</td>
			<td>abcam</td>
			<td>ab7753</td>
			<td></td>
		</tr>
		<tr>
			<td>Cigarette</td>
			<td>Marlboro</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Claudin3</td>
			<td>immunoway</td>
			<td>YT0949</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonuclase I from bovine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td>DN25</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonuclase I from bovine pancreas</td>
			<td>Sigma</td>
			<td>DN25</td>
			<td></td>
		</tr>
		<tr>
			<td>Ham&#8217;s F-12</td>
			<td>Sigma-Aldrich</td>
			<td>N6658</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin Solution&#160;</td>
			<td>Stemcell</td>
			<td>07980</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone Stock Solution</td>
			<td>Stemcell</td>
			<td>07925</td>
			<td></td>
		</tr>
		<tr>
			<td>Mucin 5AC</td>
			<td>abcam</td>
			<td>ab212636</td>
			<td></td>
		</tr>
		<tr>
			<td>Occludin</td>
			<td>proteintech</td>
			<td>27260-1-AP</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Cytosci</td>
			<td>CBS004S-BR500</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>PneumaCult-ALI 
			Basal Medium</td>
			<td>Stemcell</td>
			<td>05002&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PneumaCult-ALI 10x Supplement</td>
			<td>Stemcell</td>
			<td>05003&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PneumaCult-ALI Maintenance Supplement</td>
			<td>Stemcell</td>
			<td>05006</td>
			<td></td>
		</tr>
		<tr>
			<td>PneumaCult-Ex Plus 50x Supplement</td>
			<td>Stemcell</td>
			<td>05042</td>
			<td></td>
		</tr>
		<tr>
			<td>PneumaCult-Ex Plus Basal Medium</td>
			<td>Stemcell</td>
			<td>05041</td>
			<td></td>
		</tr>
		<tr>
			<td>Pronase E</td>
			<td>Sigma-Aldrich</td>
			<td>P5147</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat tail collagen</td>
			<td>Corning</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Stemcell</td>
			<td>07050&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro model for studying differentiation and changes of multi-omics on murine airway epithelial cells stimulated with cigarette smoke extract

Kronisk obstruktiv lungsjukdom (KOL) tillskrivs till stor del exponering för tobaksrök. Att undersöka hur luftvägsepitelceller funktionellt anpassar sig till tobaksrök är avgörande för att förstå patogenesen av KOL. Den aktuella studien syftade till att sätta upp en in vitro-modell med hjälp av primära murina luftvägsepitelceller för att efterlikna den verkliga effekten av tobaksrök. Till skillnad från etablerade cellinjer behåller primära celler mer in vivo-liknande egenskaper, inklusive tillväxtmönster, åldrande och differentiering. Dessa celler uppvisar en känslig inflammatorisk respons och effektiv differentiering, vilket nära representerar fysiologiska tillstånd. I denna modell odlades primära murina luftvägsepitelceller under 28 dagar under ett luft-vätskegränssnitt med en optimal koncentration av cigarettrökextrakt (CSE), vilket ledde till omvandlingen av ett monolager av odifferentierade celler till ett pseudostratifierat kolonnepitel, vilket indikerar CSE-acklimatisering. Omfattande multi-omics-analyser tillämpades sedan för att belysa de mekanismer genom vilka CSE påverkar differentieringen av basala luftvägsceller. Dessa insikter ger en djupare förståelse för de cellulära processer som ligger till grund för KOL-progression som svar på exponering för tobaksrök.

Chronic obstructive pulmonary disease (COPD) is a heterogeneous lung condition with complex characteristics, while patients with COPD gradually tend to be younger1. Smoking, a primary risk factor for COPD2, has a profound impact on airway epithelial cells, which serve as the initial barrier against tobacco smoke. Despite this known association, the detailed mechanisms through which tobacco smoke induces changes in airway epithelial cells remain inadequately explored. A thorough understanding of these molecular alterations is essential for identifying early diagnostic markers and therapeutic targets for COPD.
To address this gap, we developed a novel in vitro model using murine airway epithelial cells. These cells were subjected to long-term stimulation with cigarette smoke extract (CSE), enabling us to monitor dynamic cellular changes and explore the changes in airway epithelial cells under long-term tobacco stimulation and the underlying mechanism. In this model, transwell was used to provide the air-liquid interface of airway epithelial cells, and cigarette smoke extract was stimulated in the early stage of epithelial cell differentiation until the end of differentiation at 28 days. Previous studies investigated only the differentiation and short-term stimulation of airway epithelial cells (cell line dominance). They were limited to a single regulatory pathway3,4,5,6. However, the protocol presented here uses primary mouse airway epithelial cells and optimizes the cell extraction process for better cell activity than previous airway epithelial cell culture methods. This model focuses on alterations in cellular differentiation alongside comprehensive transcriptomic, proteomic, metabolomic, and epigenomic analyses. Employing immunofluorescence and advanced multi-omics techniques, we aimed to elucidate the cellular responses of airway epithelial cells to chronic tobacco smoke exposure, thereby contributing to a deeper understanding of COPD pathogenesis. This model can be used to explore the changes in airway epithelial cell differentiation pattern and its mechanism caused by long-term stimulation of various pollutants.

Författare i fokus: Utveckling och karakterisering av en in vitro-modell för att studera kronisk exponering för cigarettrök och dess inverkan på luftvägsepitelceller i KOL-forskning

In vitro-modell för att studera differentiering och förändringar av multi-omics på murina luftvägsepitelceller stimulerade med cigarettrökextrakt

The scope of our research is to develop and characterize an in vitro model using airway epithelial cells to study the effects of chronic CSC exposure. We aimed to understand the cellular response that occurred in airway epithelial cells under long-term cigarette smoke exposure, which is relevant to the pathogenesis of COPD. The recognition of intricate epithelium immune cell interaction in COPD is considered a pivotal recent development in COPD research.We have established a novel in vitro model that mimic chronic CSC exposure in airway epithelial cells. Our findings open up new avenue of investigating the long-term effect of chronic cigarette smoke exposure on airway epithelial cells and their role in COPD progression. In the future, our laboratory will focus on exploring specific pathway that regulate epithelial cells differentiation and the role of airway epithelial cells heterogenetic in COPD treatment response.

Differentiering Murina luftvägsepitelceller differentierades framgångsrikt efter odling vid ett luft-vätskegränssnitt med ett differentieringsmedium i 28 dagar. Förekomst av cilierade celler och bägarceller påvisades genom immunofluorescensanalys av ciliemarköracetylerat α-Tubulin (grönt; Figur 3A) och bägarcellsmarkören Mucin5AC, respektive6 (röd; figur 3B).
Bestämning av CSE-koncentration f?...

Här beskriver vi en in vitro-modell för att isolera och differentiera murina luftvägsepitelceller, med fokus på deras acklimatisering till kroniskt cigarettrökextrakt (CSE). Modellen kan användas för att på ett heltäckande sätt karakterisera multi-omics-effekten av CSE, vilket möjligen ger insikter i de cellulära svaren under kronisk rökexponering.

Chronic obstructive pulmonary disease (COPD) is largely attributed to tobacco smoke exposure. Investigating how airway epithelial cells functionally adapt ...

in-vitro-model-for-studying-differentiation-changes-multi-omics-on

Research

JoVE Journal

Biology

In Vitro Model for Studying Differentiation and Changes of Multi-Omics on Murine Airway Epithelial Cells Stimulated with Cigarette Smoke Extract

Chronic obstructive pulmonary disease (COPD) is largely attributed to tobacco smoke exposure. Investigating how airway epithelial cells functionally adapt to tobacco smoke is crucial for understanding the pathogenesis of COPD. The present study was to set up an in vitro model using primary murine airway epithelial cells to mimic the real-life impact of tobacco smoke. Unlike established cell lines, primary cells retain more in vivo-like properties, including growth patterns, aging, and differentiation. These cells exhibit a sensitive inflammatory response and efficient differentiation, thus closely representing physiological conditions. In this model, primary murine airway epithelial cells were cultured for 28 days under an air-liquid interface with an optimal concentration of cigarette smoke extract (CSE), which led to the transformation of a monolayer of undifferentiated cells into a pseudostratified columnar epithelium, indicative of CSE acclimation. Comprehensive multi-omics analyses were then applied to elucidate the mechanisms by which CSE influences the differentiation of basal airway cells. These insights provide a deeper understanding of the cellular processes underpinning COPD progression in response to tobacco smoke exposure.

Here, we describe an in vitro model for isolating and differentiating murine airway epithelial cells, focusing on their acclimation to chronic cigarette smoke extract (CSE). The model could be utilized to comprehensively characterize the multi-omics impact of CSE, which possibly provides insights into the cellular responses under chronic smoke exposure.

Isolation of Primary Murine Airway Epithelial Cells from Murine Tracheas

To begin, arrange freshly prepared expansion medium, filter sterilized proteinase and antibiotic solutions, and sterilized surgical instruments on the working platform of the biosafety cabinet. Add rat tail collagen into 100-millimeter culture dishes and 24-millimeter trans wells. Leave the dishes open overnight under ultraviolet light for sterilization.Immerse the euthanized mouse in 75%ethanol for sterilization. Do not immerse the nose and mouth. Using surgical scissors and forceps, cut and open the epidermis from the lower jaw to the abdominal cavity of the mouse.Using another set of surgical scissors and forceps, tear apart the thyroid glands on both sides and remove the connections between the trachea, the surrounding muscle tissue, and the esophagus. Next, carefully cut into the chest, and use forceps to delve deeper into the chest cavity. Remove the entire lung and find the end of the trachea.Cut out the trachea from the thyroid cartilage to the tracheal bronchial branch, and put it in a pre cold expansion medium containing four antibiotics. Shake the tube to rinse as much blood as possible off the surface of the trachea before placing it on ice. Now transfer the trachea to pre cold PBS containing four antibiotics.Using surgical scissors and forceps, remove clots and other tissue from the surface of the trachea and cut them into one square centimeter size. Incubate tracheal tissue in proteinase solution at 37 degrees Celsius. After 40 minutes, rock the tube several times and use a 40-micron cell strainer to remove the remaining tissues.Rinse the chopped trachea on a strainer with five milliliters of expansion medium. Centrifuge the tracheal suspension at 400 G for five minutes at four degrees Celsius, and resuspend the pellet in eight milliliters of expansion medium. Count the viable cells using trypan blue and the hemocytometer.Plate P0 cell suspension on a 100-millimeter dish, precoated with rat tail collagen. Culture the cells at 37 degrees Celsius with 5%carbon dioxide.

Isolering av primära murina luftvägsepitelceller från murina luftstrupar

Expansion Culture and Passage of Primary Murine Airway Epithelial Cells

Once the murine tracheal epithelial cells form an intact monolayer with a cobblestone appearance, gently rinse the cells with three milliliters of prewarmed PBS. Add three milliliters of animal component-free enzymatic dissociation solution to the dish and incubate at 37 degrees Celsius for five minutes. Gently pipette to dislodge the cells and transfer the suspension to three milliliters of animal component-free enzyme inhibition solution.Centrifuge the cell suspension at 400 G for five minutes at four degrees Celsius and resuspend the pellet in one milliliter of expansion medium. Count the viable cells using trypan blue and a hemocytometer. Plate P1 cell suspension on a 100 millimeter dish pre-coated with rat tail collagen.Culture the cells at 37 degrees Celsius with 5%carbon dioxide.

Expansionsodling och passage av primära murina luftvägsepitelceller

Differentiation and Cigarette Smoke Extract (CSE) Stimulation of Primary Murine Airway Epithelial Cells at Air-Liquid Interface

Once the primary murine airway epithelial cells reach 80%confluency, seat cells on a glass slide coated with rat tail collagen. Then, add paraformaldehyde to fix the cells for 12 hours. Wash the slide three times with PBS for five minutes each, and incubate it with 3%bovine serum albumin and 0.1%Triton X-100 in PBS for one hour.Add anti-pan cytokeratin antibody at a dilution of one to 500 onto the slide, and incubate overnight at four degrees Celsius. The next day, wash the slide three times with PBS solution for five minutes each before incubating with secondary antibody at a dilution of one to 1, 000 for two hours in the dark. After washing the slide with PBS, add DAPI at a dilution of one to 1, 000 and incubate for 15 minutes.Wash the slide once with PBS for five minutes. Observe the slide under a fluorescence microscope to compare expression patterns to a positive control and a negative control After detachment and centrifugation, re-suspend the P2 phase cells in an appropriate volume of expansion medium. Pipette one milliliter of cell suspension onto the apical chamber of the Transwell polycarbonate membrane insert.Add 1.5 milliliters of proliferation media to the basal compartment of the Transwell. Incubate the cells at 37 degrees Celsius until confluence is reached. Prepare 50 milliliters of complete differentiation medium using the following components.Bubble one cigarette through 12.5 milliliters of differentiation medium, and then filter it through a 0.22 micron pour filter to prepare cigarette smoke extract or CSE. To ensure standardization between experiments and batches of CSE, measure the absorbance at 320 nanometers on a spectrophotometer. Next, remove the expansion medium from the apical and basal chamber of Transwells.Add a differentiation medium containing an appropriate concentration of CSE to stimulate primary murine airway epithelial cells for 28 days. During incubation, wash the apical chamber twice a week with PBS and replace the medium in the basal chamber with freshly prepared differentiation medium containing CSE. Murine airway epithelial cells successfully differentiated at an air liquid interface in 28 days.The presence of ciliated and goblet cells was demonstrated by immunofluorescence assay of cilia marker, acetylated alpha tubulin, and the goblet cell marker mucin 5AC respectively. Cell viability at 24 hours with varying CSE concentrations demonstrated that epithelial cells declined when the concentration was higher than 6%Cell activity under long-term stimulation showed no significant cell death with 2%4%and 6%CSE concentrations. But changes in transmembrane resistance and exfoliated cells were noted.In addition, an overall reduction in differentiated cells and ciliated cells was noted when murine airway epithelial cell cultures were chronically exposed to CSE.