המחברים מבקשים להודות למוריס סמית &#39;ומריה Talikka לעיונם של כתב היד.

<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=The+forces+applied+by+cilia+depend+linearly+on+their+frequency+due+to+constant+geometry+of+the+effective+stroke.">The forces applied by cilia depend linearly on their frequency due to constant geometry of the effective stroke.</a> Biophysical journa. 94, 298-305 (2008).</li><li>Hilgendorf, C., 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=Caco-2+versus+Caco-2/HT29-MTX+co-cultured+cell+lines:+permeabilities+via+diffusion,+inside-+and+outside-directed+carrier-mediated+transport.">Caco-2 versus Caco-2/HT29-MTX co-cultured cell lines: permeabilities via diffusion, inside- and outside-directed carrier-mediated transport.</a> J Pharm Sc. 89, 63-75 (2000).</li><li>Sutto, Z., Conner, G. 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מחקר זה מומן על ידי פיליפ מוריס אינטרנשיונל.

הנה, יש לנו הפגנו את תחולתו של הסימפונות organotypic אדם ודגמי רקמת האף על מנת להעריך את ההשפעה של חשיפה חוזרת ונשנית CS. כחלופה לניסויים בבעלי חיים, מספר מערכות חשיפה פותחו להערכות טוקסיקולוגית חשיפת תרסיס במבחנה (לדוגמא., Vitrocell, Cultex, אליס, וכו &#39;). מודולים חשיפה אלה יכולים גם להיות מנוצלים להערכה טוקסיקולוגית של מזהמים באוויר, חלקיקים הנישאים באוויר, חלקיקים, וכו &#39;במחקר זה, השתמשנו במערכת Vitrocell שיכולים להכיל עד 48 מדגמים שונים בו זמנית, מה שמאפשר לניסויים בקנה מידה גדולים יותר והשתנות נמוכה יותר בין טיפולים. לכל חשיפת תרסיס במבחנה, את הסיכון של זיהום בתרבית הרקמה נשאר סיכון עיקרי להפחתת סיכון שדורש טיפול זהיר של תרביות רקמה בכל הניסויים. מדידת חלקיקים בתצהיר בזמן אמת על microbalance במהלך הדוארניסוי xposure מאפשר לפקח שמינוני CS שנוצרו ממערכת החשיפה העומדים בציפיות. כדי להבטיח את הדיוק של החלקיקים נמדד בתצהיר, הקמה של מדידת האינטרנט בצורה נכונה לפני החשיפה הוא critial, למשל, הגדרת קנה המידה ל0. בנוסף, בגלל ההבדל בין תחומי microbalance (2 סנטימטר) ו הוספת תרבית רקמה (0.33 סנטימטר 2), ושינינו את החישוב הסופי לאזור של להכניס את התרבות: רק 33% מהתצהיר על microbalance משקפים את התצהיר בפועל בכנס התרבות. מדידה של Teer לברר פונקציונלי מחסום הדוק צומת ולהעריך שיבוש שכבת האפיתל היא הליך קל יחסית ליישום, שבו אנו מדווחים כאן. עם זאת, מכיוון שמודלי רקמת הסימפונות ואף מכילים תאי גביע ובין מייצרי ריר, כביסה הפסגה צריכה להתבצע לפני שקילת משקל הג Teerurement. הכביסה הפסגה היא קריטית משום שהנוכחות של שכבת הריר וההשתנות של ההטיה פח עובי שלה המדידה של Teer, interferring עם ההשפעה של חשיפת CS. רעיון זה הוא בהסכם עם מה שדווח על ידי Hilgendorf ועמיתים, שבי החדירות של קאקו-2 תאים הושפעו משיתוף culturing עם שורת תאי גביע מייצרי ריר HT29 23. הריר צריך לשטוף לפני מדידת Teer, כי כביסה הפסגה ממש לפני החשיפה עלולה להפריע לתגובות הרקמות לCS. לכן, המדידה מתבצעת שלושה ימים לפני החשיפה, ולא ממש לפני החשיפה. הראינו כי פעילות CYP1A1 / CYP1B1 ניתן הייתה למדוד מהמודלים תרבות organotypic בעקבות חשיפת CS למרות הפעילות מוגברת בצניעות רק על ידי CS. אות חלשה זה יכול להיות מוגבר על ידי דגירה ארוכה יותר של מצע CYP1A1 / 1B1 (כלומר., luciferin-CEE), למשל ל24 שעות (מידע לא מוצג). אחת המגבלות של מדידת פעילות CYP בעבודה הנוכחית הוא היעדר הנורמליזציה לרמת חלבון CYP או לספירת התאים, אשר יכול להילקח בחשבון למחקרים עתידיים כדי להבטיח שהשינוי בפעילות האנזים אינו מושפע על ידי השינוי של שתי רמת החלבון או ספירת התאים. אנחנו דיווחו כי חשיפת CS בלמה את הריסים להכות בשני האף ודגמי רקמת הסימפונות. תצפיות דומות נעשו במודלים של יונקים ושאינם יונקים מגוונים 12. לריסים להכות מדידה, על מנת להבטיח כי רקמות מטופלות ומטופלים באופן דומה הוא קריטי, לדוגמא, אם שינוי בינוני מיושם, זה צריך להיות מיושם על כל הדגימות. Sutto ועמיתים דיווחו כי pH השפיע על ריסי היונקים להכות תדירות 24. לפיכך, בעת השוואה בין מכות תדרי תאים שטופלו בcompunds / תערובות שונות, pHהתאמה צריכה להיחשב כדי למזער את ההשתנות של מדידת מכות הריסים. יתר על כן, בטמפרטורה שבי המדידה מתבצעת היא קריטית גם כתדר של מכות ריסי טיפות עם ירידה בטמפרטורה. כדי למזער את השונות כתוצאה מהשינויים אלה, חממת במה עליונה, מצוידים בטמפרטורה, CO 2, ובקרת לחות שימש במחקר זה. למרות זאת, שמו לב שהריסים להכות תדרים ברקמות הסימפונות לאחר חשיפת CS היו משתנים מאוד (כלומר., להגדיל בעלון אחד ולהקטין בעלון האחר), טוענים כי מכות הריסים נכונה מופרעים מאוד לאחר חשיפה. בניגוד לכך, אנו נצפו היעדר תדרי מכות הריסים מדידים ברקמת האף לאחר חשיפת CS, המצביע על כך התגובה של רקמות האף היא יותר רגישה ועקבית. זה בהסכם עם פרסום קודם שהראה כי יש לו את רקמות האף קיבולת נמוכה כדי לטהר כלעומת הסמפונות 25. לבסוף, הראינו כי ביטוי גני פרופיל מסימפונות organotypic ודגמי רקמת האף נחשפו לCS יכול להדגים השפעה של CS על חילוף חומרים xenobiotic. מעניין לציין, כי השינוי שנצפה בחילוף חומרי xenobiotic בסימפונות organotypic והאף במודלים חוץ גופית החשוף לCS דומה לזה של מצב in vivo במעשנים כפי שנדונו בפירוט רב יותר בפרסום קודם 15. לביטוי גני ניתוחים, שימוש במכשיר רובוטי עושה ניתוח תפוקה גבוהה אפשרי. יתר על כן, טיפול רובוטית אוטומטי מגדיל עוד יותר את העקביות ודיוק של תוצאות ביטוי גנים. עם זאת, אוסף מהיר של דגימות הרקמה היה קריטי כדי למנוע השפלה RNA במהלך הפקת RNA. עיבוד RNA וגישות transcriptomic המתואר כאן יכולים להיות מיושמים גם לדגימות רקמת in vivo.

Lungs are directly and constantly exposed to inhaled air that may contain diverse toxicants such as pollutants and constituents of cigarette smoke (CS). Studying the impact of exposure to those toxicants on respiratory tissues is most informative when done in a manner that resembles in vivo exposure. Compared with the classical 2D immersed cell cultures (e.g., normal human bronchial epithelial cells (NHBE)), 3D organotypic tissue models better recapitulate the morphological, physiological, and molecular aspects of the human airway epithelium in vivo1,2: the 3D tissue models contain the diversity of the cell types observed in vivo, including differentiated epithelial cells, ciliated and non-ciliated cells, goblet cells, and basal cells. They have functional tight junctions and exhibit a mucociliary phenotype 1-3. Moreover, the cultures can be grown on a permeable porous membrane, in an air-liquid interface, allowing a direct exposure to aerosol at the apical side (whereas the basolateral side is immersed in culture medium) 3-5. Dvorak and colleagues reported that gene expression profiles of bronchial tissue models were similar to those obtained from human bronchial brushings 3. In addition, Mathis and colleagues showed that the responses of these tissue models to CS were similar to the differences observed between bronchial epithelial cells obtained from smokers and cells obtained from non-smokers 6. Finally, because the bronchial tissue models could be cultured for up to several months 4,5, they could potentially be used to examine the effects of long-term exposure of test items. 
Cytotoxicity assessments are common parameters measured following chemical insults or to assess the toxicity of specific compounds or mixtures. For instance, membrane integrity can be measured by a luminescent assay and allows the measurement of a dose-dependent cytotoxic effect on the cell culture 7. However, to assess pathophysiological effects of compounds at subtoxic concentrations, other parameters should be measured. For example, tissue integrity determined using the transepithelial electrical resistance (TEER) assay ensures the functionality of tight junctions and monitors the disruption of the epithelial layer 8,9. Ciliary beating frequency also allows the measurement of CS-related effects on respiratory tissues. A normal beating frequency for the cilia lining bordering the upper and lower respiratory tract is important to protect against airway infections 10. Each of the ciliated columnar epithelial cells of the respiratory epithelium has 200-300 cilia beating at a particular frequency to eliminate infectious agents or inhaled particulate matter trapped in the mucus released by interspersed goblet cells 11. CS contains chemicals that may inhibit ciliary beating 12, leading to a reduced protection of the respiratory tract. This work shows that ciliary beating can be measured in organotypic tissue models. This approach allows assessment of whether epithelial cells exhibit their normal function in the organotypic tissue culture. CS also activates xenobiotic metabolism responses in the respiratory tract to metabolize tobacco smoke constituents 13. The activity of the phase I xenobiotic metabolism enzymes, CYP1A1 and CYP1B1, of the tissue models can be measured. Additionally, as previously reported, global gene expression can be measured in the organotypic bronchial tissue models 6,14,15. A transcriptomic data and network-based systems biology approach is leveraged to assess the impact of CS on xenobiotic metabolism 15. 
The methodologies used to expose organotypic 3D bronchial and nasal tissue models to mainstream CS using an in vitro exposure system and to measure the tissue responses to this exposure compared to fresh air exposure (control) are detailed here.

<table border="0" cellpadding="0" cellspacing="0" width="1545">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">Name of Material/ Equipment</td>
			<td style="width:367px;">Company</td>
			<td style="width:655px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MucilAir-human fibroblasts-bronchia</td>
			<td>Epithelix S&#225;rl, Geneva, Switzerland</td>
			<td>http://www.epithelix.com/content/view/122/19/lang,en/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MucilAir Culture Medium</td>
			<td>Epithelix S&#225;rl, Geneva, Switzerland</td>
			<td>http://www.epithelix.com/content/view/84/16/lang,en/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VITROCELL</td>
			<td>VITROCELL systems GmbH, Waldkirch, Germany</td>
			<td>http://www.vitrocell.com/index.php?Nav_Nummer=2&#38;R=</td>
			<td></td>
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			<td>University of Kentucky</td>
			<td>http://www2.ca.uky.edu/refcig/</td>
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			<td>La Haute &#201;cole de Gestion (HESGE), Geneva, Switzlerland</td>
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			<td>Leica, Heerbrugg, Switzerland</td>
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			<td>World Precision Instruments</td>
			<td>http://www.wpiinc.com/products/physiology/stx2-chopstick-electrode-set-for-evom2/</td>
			<td></td>
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			<td>World Precision Instruments</td>
			<td>http://www.wpiinc.com/products/physiology/evom2-evom2-epithelial-voltohmmeter-for-teer/</td>
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			<td>Roche</td>
			<td>http://www.roche.com/products/product-details.htm?region=us&#38;type=product&#38;id=66</td>
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			<td>http://www.sigmaaldrich.com/catalog/product/sial/288306?lang=en&#38;region=</td>
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			<td>Thermo Scientific</td>
			<td>http://www.nanodrop.com/</td>
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			<td>http://www.genomics.agilent.com/en/Bioanalyzer-System/2100-Bioanalyzer-Instruments/?cid=AG-PT-106</td>
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			<td>Affymetrix</td>
			<td>http://www.affymetrix.com/catalog/prod370001/AFFY/High-Throughput-(HT)-Whole-Transcript-(WT)-Kit</td>
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			<td>Affymetrix</td>
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</table>

impact assessment of repeated exposure of organotypic 3d bronchial and nasal tissue culture models to whole cigarette smoke

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease or lung cancer. To understand the underlying mechanisms of disease development, it would be important to examine the impact of CS exposure directly on lung tissues. However, this approach is difficult to implement in epidemiological studies because lung tissue sampling is complex and invasive. Alternatively, tissue culture models can facilitate the assessment of exposure impacts on the lung tissue. Submerged 2D cell cultures, such as normal human bronchial epithelial (NHBE) cell cultures, have traditionally been used for this purpose. However, they cannot be exposed directly to smoke in a similar manner to the in vivo exposure situation. Recently developed 3D tissue culture models better reflect the in vivo situation because they can be cultured at the air-liquid interface (ALI). Their basal sides are immersed in the culture medium; whereas, their apical sides are exposed to air. Moreover, organotypic tissue cultures that contain different type of cells, better represent the physiology of the tissue in vivo. In this work, the utilization of an in vitro exposure system to expose human organotypic bronchial and nasal tissue models to mainstream CS is demonstrated. Ciliary beating frequency and the activity of cytochrome P450s (CYP) 1A1/1B1 were measured to assess functional impacts of CS on the tissues. Furthermore, to examine CS-induced alterations at the molecular level, gene expression profiles were generated from the tissues following exposure. A slight increase in CYP1A1/1B1 activity was observed in CS-exposed tissues compared with air-exposed tissues. A network-and transcriptomics-based systems biology approach was sufficiently robust to demonstrate CS-induced alterations of xenobiotic metabolism that were similar to those observed in the bronchial and nasal epithelial cells obtained from smokers.

בתוצאות שתתוארנה להלן, רקמות organotypic היו תאים ראשוניים אדם אפיתל מבודד מעישון בריא,, תורמי קווקזי והיו מחדש באמצעות fibroblasts 15. הסימפונות 3D ודגמי רקמת האף בסימפונות מבחנה ומודלי organotypic האף דומה ברמה התאית אפיתל בדרכי הנשימה האנושי (איור 2). חשיפת CS הסימפונות Organotypic ודגמי רקמת האף יכולים להיות חשופים שוב ושוב לCS הזרם המרכזי בממשק האוויר הנוזלי. ההליך הניסיון החשיפה לעישון משמש לתוצאות מאוירות כאן מתואר באיור 1. הקלטה של מכות הריסים מכות cilia נרשמו ברקמות חשופות האוויר כפי שמודגם בקטעי הווידאו. חשיפת CS הייתה קשורה בהפחתת תדירות מכות הריסים: השיא החזק שנצפה סביב4 הרץ בדמה החשוף ולפני החשיפה לא נצפה יותר לאחר חשיפת CS (איור 3). תדירות מופחתת זה תגרום cilia להכות פחות יעיל להסרת ריר וחומרים מזהמים 22. מדידה של Teer וCYP1A1 / פעילות 1B1 Teer נמדד 48 שעות לאחר החשיפה האחרונה כדי להבטיח את הרקמה עדיין פונקציונלית. CYP1A1 / 1B1 פעילות גם נמדדה 48 שעות לאחר תום החשיפה כפי שמודגמת באיור 1. ערכי Teer ברקמות חשופות-CS לא היו שונים באופן משמעותי בהשוואה לרקמות חשופות אוויר המאשר כי אין השפעה רעילה לתאים גדולה של עשן בריכוז זה. שבלאחר החשיפה 48 שעות, CYP1A1 / 1B1 פעילות של הרקמות הייתה מעט מוגברת, המצביעה על עלייה צנועה של מנגנון הגנת רקמות בעקבות החשיפה (איור 4). דו פרופילי ביטוי ומערכות מבוססי רשת גן<html> גישת ology ללמוד את ההשפעה של חשיפת CS על השינוי של חילוף חומרים xenobiotic רקמות נאספו בנקודות זמן לאחר חשיפה 48 שעות. בהמשך לכך, ניתוח microarray נערך כדי ליצור פרופילי ביטוי גנים של CS- ורקמות חשוף אוויר. השפעה של חשיפת CS על חילוף חומרים xenobiotic נחקרה תוך שימוש בגישה ביולוגיה מערכות מבוסס רשת ממונפת מפרופילי גן-ביטוי וכפי שדווח קודם לכן 15. שימוש בגישה ביולוגיה מערכות מבוסס רשת מראה כי השפעות חשיפת CS ברמה של צמתים עמוד השדרה במודל רשת מטבוליזם Xenobiotic היו בקורלציה יפה בין במבחנה ובמערכי נתוני vivo (איור 5). בניגוד לכך, ברמה של כל שינויים גנטיים-ביטוי אישי, מתאמים עניים נצפו בין במבחנה (CS-חשוף מול חשוף אוויר) וin vivo (מעשנים לעומת הלא מעשנים). r.within-page = &quot;תמיד&quot;&gt; <img alt="איור 1" src="/files/ftp_upload/52325/52325fig1.jpg" /> איור 1. הליך סכמטי של החשיפה החוזרת ונשנית של CS לדגמי רקמת הסימפונות organotypic קיצורים:. CYP, P450s ציטוכרום; CS, עשן סיגריות; Teer, התנגדות חשמלית transepithelial. <img alt="איור 2" src="/files/ftp_upload/52325/52325fig2.jpg" /> איור 2. Organotypic הסימפונות ודגמי תרבית רקמת האפיתל האף. Cartoon הממחיש את הסימפונות () ולהכניס את תרבות האף רקמה (B) בממשק האוויר נוזלי. המודלים במבחנה הכילו תאי ריסי שמוצגים בשכבת הפסגה של התאים המוכתמים Hematoxylin וEosin (C לסימפונות, D לאף) כפי שדווחו בעבר 15. הדגמים היו שיתוף תרבותי עם fibroblasts שחשוב<html> לצמיחה וההתמיינות של תאי אפיתל (מסומן בחצים). הכתמה של סמני שושלת דרכי הנשימה: p63 (E לסימפונות, F לאף) וMuc5AC (G לסימפונות, H לאף) מוצג כפי שדווח בעבר 15. <a href="https://www.jove.com/files/ftp_upload/52325/52325fig2large.jpg" target="_blank">אנא לחץ כאן כדי לצפות בגרסה גדולה יותר של דמות זו.</a> <img alt="איור 3" src="/files/ftp_upload/52325/52325fig3.jpg" /> תדירות איור 3. צילה תדר מכות. צילה מכות (CBF) נמדדה בחשוף אוויר ובתרבות לפני ואחרי החשיפה. בירידה בCBF נתפס לאחר חשיפת CS (תוצאות נציג משני להכניס מוצגות כלשכפל 1 (R1) ולשכפל 2 (R2)). מודעה / 52,325 / 52325fig4.jpg &quot;/&gt; מדידת איור 4. CYP1A1 / פעילות CYP1B1 וTeer. Pannels השמאל מצביע על פעילות CYP1A1 / CYP1B1 בסימפונות () ודגמי רקמת האף (B). לוחות מימין מצביעים על מדידת Teer בסימפונות (C) ודגמי רקמת האף (D). אמצעי ± SD מוצג. קיצורים: CS, עשן סיגריות; CYP, ציטוכרום P450; Teer, התנגדות חשמלית transepithelial; RLU, יחידת luminescense היחסית. <img alt="איור 5" src="/files/ftp_upload/52325/52325fig5.jpg" /> איור 5. גישת מערכות מבוססת רשת ביולוגיה להערכת ההשפעה של חשיפת CS בהקשר של חילוף חומרים xenobiotic. השינויים ברמות ביטוי הגן שמשו לחישוב ההפעלה של צומת עמוד השדרה של מודל רשת Xenobiotic מטבוליזם () </<html> strong&gt; כפי שדווח בפרסום קודם 15. הדמות מהימין ממחישה את הרעיון של כימות של בלוטות עמוד השדרה, המכונות גם &quot;ערך עמוד השדרה רשת ההפרש,&quot; בגישת רשות הטבע והגנים. האליפסות הכחולות מייצגות את הפעילות של בלוטות עמוד השדרה (כלומר., השכבה הפונקציונלית) והכדורים הירוקים מייצגים את הביטוי של גנים (כלומר., שכבת תעתיק). השפעות של חשיפת CS ברקמות הסימפונות organotypic (במבחנה, CS-חשופים מול חשוף אוויר) בשעות 48 של postexposure הושווה לאלה של עישון על אפיתל הסימפונות האנושי שנאסף על ידי אפיתל ברונכוסקופיה והאף על ידי burshing חלזוני הנחות (GSE16008 ) (In vivo, מעשנים לעומת הלא מעשנים) (B). ריבועים, מתאם של ביטוי גנים משתנים בין במבחנה ובמערכי נתוני vivo. קיצורים: CS, עשן סיגריות; רשות טבע וגנים, משרעת ההפרעות רשת.&quot;Target =&quot; ove.com/files/ftp_upload/52325/52325fig5large.jpg _ blank &quot;&gt; לחץ כאן כדי לצפות בגרסה גדולה יותר של דמות זו.

Watch this Scientific Journal Video about Impact Assessment of Repeated Exposure of Organotypic 3D Bronchial and Nasal Tissue Culture Models to Whole Cigarette Smoke at JoVE.com

Impact Assessment of Repeated Exposure of Organotypic 3D Bronchial and Nasal Tissue Culture Models to Whole Cigarette Smoke

The aim of this protocol is to expose human organotypic 3D bronchial and nasal tissue models to mainstream cigarette smoke (CS) at the air-liquid interface. The impact of CS on the tissues is then investigated using a cytochrome P450 activity assay, a cilia beating measurement, and a systems biology approach.

הערכת השפעה של חשיפה חוזרת ונשנית של דוגמניות תרבות רקמות Organotypic 3D הסימפונות ובאף לעשן סיגריות שלמות

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease ...

impact-assessment-repeated-exposure-organotypic-3d-bronchial-nasal

Research

JoVE Journal

Bioengineering

11.0K Views.  Philip Morris Products S.A.. The aim of this protocol is to expose human organotypic 3D bronchial and nasal tissue models to mainstream cigarette smoke (CS) at the air-liquid interface. The impact of CS on the tissues is then investigated using a cytochrome P450 activity assay, a cilia beating measurement, and a systems biology approach.

Video: Impact Assessment of Repeated Exposure of Organotypic 3D Bronchial and Nasal Tissue Culture Models to Whole Cigarette Smoke

A Protocol for Detecting and Scavenging Gas-phase Free Radicals in Mainstream Cigarette Smoke

Cigarette smoking is associated with human cancers. It has been reported that most of the lung cancer deaths are caused by cigarette smoking 5,6,7,12. Although tobacco tars and related products in the particle phase of cigarette smoke are major causes of carcinogenic and mutagenic related diseases, cigarette smoke contains significant amounts of free radicals that are also considered as an important group of carcinogens9,10. Free radicals attack cell constituents by damaging protein structure, lipids and DNA sequences and increase the risks of developing various types of cancers. Inhaled radicals produce adducts that contribute to many of the negative health effects of tobacco smoke in the lung3. Studies have been conducted to reduce free radicals in cigarette smoke to decrease risks of the smoking-induced damage. It has been reported that haemoglobin and heme-containing compounds could partially scavenge nitric oxide, reactive oxidants and carcinogenic volatile nitrosocompounds of cigarette smoke4. A 'bio-filter' consisted of haemoglobin and activated carbon was used to scavenge the free radicals and to remove up to 90% of the free radicals from cigarette smoke14. However, due to the cost-ineffectiveness, it has not been successfully commercialized. Another study showed good scavenging efficiency of shikonin, a component of Chinese herbal medicine8. In the present study, we report a protocol for introducing common natural antioxidant extracts into the cigarette filter for scavenging gas phase free radicals in cigarette smoke and measurement of the scavenge effect on gas phase free radicals in mainstream cigarette smoke (MCS) using spin-trapping Electron Spin Resonance (ESR) Spectroscopy1,2,14. We showed high scavenging capacity of lycopene and grape seed extract which could point to their future application in cigarette filters. An important advantage of these prospective scavengers is that they can be obtained in large quantities from byproducts of tomato or wine industry respectively11,13

Spin-trapping ESR spectroscopy was used to study the effect of plant antioxidants lycopene, pycnogenol and grape seed extract on scavenging gas-phase free radicals in cigarette smoke.

פרוטוקול לאיתור גז הדחה בשלב רדיקלים חופשיים עשן סיגריות רגילה

Cigarette smoking is associated with human cancers. It has been reported that most of the lung cancer deaths are caused by cigarette smoking 5,6,7,12.  ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

הנדסת רקמות של 3D אדם<em&gt; במבחנה</emמערכת מבחן גידול&gt;

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

לדוגמא הכנת אסטרטגיות להדמית ספקטרומטריית המסה של מודלי תרבית תאי 3D

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioprinting של הסחוס, תחליפי רקמת העור ניצול פסיבים הרומן ערבוב טכניקה יחידה עבור Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

A Microcontroller Operated Device for the Generation of Liquid Extracts from Conventional Cigarette Smoke and Electronic Cigarette Aerosol

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among adults. High quality, reproducible research on the consequences of electronic cigarette use is essential for understanding emerging public health concerns and crafting evidence based regulatory policy. While a growing number of papers discuss electronic cigarettes, there is little consistency in methods across groups and very little consensus on results. Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This protocol details instructions for the assembly and operation of said device, and demonstrates the use of the generated extract in two sample applications: an in vitro cell viability assay and gas-chromatography mass-spectrometry. This method provides a tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This method provides a useful tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

מיקרו-בקר המופעל התקן לדור של נוזלי תמציות קונבנציונאלי סיגריה סיגריה עשן ואלקטרוניקה תרסיס

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

דגמי כבד תלת-ממדיים מתקדמים לבדיקות גנוטוקסיות במבחנה בעקבות חשיפה ננו-חומרית לטווח ארוך

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

An Open Source Technology Platform to Manufacture Hydrogel-Based 3D Culture Models in an Automated and Standardized Fashion

Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These issues represent drawbacks in workflows that can ultimately result in irreproducibility of research findings. Manual-based workflows are further limiting the advancement and widespread adoption of viscous materials, such as hydrogels used for biomedical applications. These challenges can be overcome by using automated workflows with standardized mixing processes to increase reproducibility. In this study, we present step-by-step instructions to use an open source protocol designer, to operate an open source workstation, and to identify reproducible mixtures. Specifically, the open source protocol designer guides the user through the experimental parameter selection and generates a ready-to-use protocol code to operate the workstation. This workstation is optimized for pipetting of viscous materials to enable automated and highly reliable handling by the integration of temperature docks for thermoresponsive materials, positive displacement pipettes for viscous materials, and an optional tip touch dock to remove excess material from the pipette tip. The validation and verification of mixtures are performed by a fast and inexpensive absorbance measurement of Orange G. This protocol presents results to obtain 80% (v/v) glycerol mixtures, a dilution series for gelatin methacryloyl (GelMA), and double network hydrogels of 5% (w/v) GelMA and 2% (w/v) alginate. A troubleshooting guide is included to support users with protocol adoption. The described workflow can be broadly applied to a number of viscous materials to generate user-defined concentrations in an automated fashion.

This protocol serves as a comprehensive tutorial for standardized and reproducible mixing of viscous materials with a novel open source automation technology. Detailed instructions are provided on the operation of a newly developed open source workstation, the usage of an open source protocol designer, and the validation and verification to identify reproducible mixtures.

פלטפורמת טכנולוגיית קוד פתוח לייצור דגמי תרבות תלת-ממדית מבוססי הידרוג'ל באופן אוטומטי ומתוקנן

Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These ...

Isolation and Culture of Primary Human Gingival Epithelial Cells using Y-27632

The gingival tissue is the first structure that protects periodontal tissues and plays meaningful roles in many oral functions. The gingival epithelium is an important structure of gingival tissue, especially in the repair and regeneration of periodontal tissue. Studying the functions of gingival epithelial cells has crucial scientific value, such as repairing oral defects and detecting the compatibility of biomaterials. As human gingival epithelial cells are highly differentiated keratinized cells, their lifespan is short, and they are difficult to passage. So far, there are only two ways to isolate and culture gingival epithelial cells, a direct explant method and an enzymatic method. However, the time required to obtain epithelial cells using the direct explant method is longer, and the cell survival rate of the enzymatic method is lower. Clinically, the acquisition of gingival tissue is limited, so a stable, efficient, and simple in vitro isolation and culture system is needed. We improved the traditional enzymatic method by adding Y-27632, a Rho-associated kinase (ROCK) inhibitor, which can selectively promote the growth of epithelial cells. Our modified enzymatic method simplifies the steps of the traditional enzymatic method and increases the efficiency of culturing epithelial cells, which has significant advantages over the direct explant method and the enzymatic method.

Here we present a modified method for the isolation and culture of human gingival epithelial cells by adding the Rock inhibitor, Y-27632, to the traditional method. This method is easier, less time-consuming, enhances stem cell properties, and produces larger numbers of high-potential epithelial cells both for the laboratory and for clinical applications.

בידוד ותרבות של תאי אפיתל חניך אנושיים ראשוניים באמצעות Y-27632

The gingival tissue is the first structure that protects periodontal tissues and plays meaningful roles in many oral functions. The gingival epithelium is ...

ייצור מדרגי של כדורי תאים אחידים באמצעות התקן הרכבה אקוסטית תלת מימדית

Three-Dimensional Acoustic Assembly Device for Mass Manufacturing of Cell Spheroids

Cell spheroids are promising three-dimensional (3D) models that have gained wide applications in many biological fields. This protocol presents a method for manufacturing high-quality and high-throughput cell spheroids using a 3D acoustic assembly device through maneuverable procedures. The acoustic assembly device consists of three lead zirconate titanate (PZT) transducers, each arranged in the X/Y/Z plane of a square polymethyl methacrylate (PMMA) chamber. This configuration enables the generation of a 3D dot-array pattern of levitated acoustic nodes (LANs) when three signals are applied. As a result, cells in the gelatin methacryloyl (GelMA) solution can be driven to the LANs, forming uniform cell aggregates in three dimensions. The GelMA solution is then UV-photocured and crosslinked to serve as a scaffold that supports the growth of cell aggregates. Finally, masses of matured spheroids are obtained and retrieved by subsequently dissolving the GelMA scaffolds under mild conditions. The proposed new 3D acoustic cell assembly device will enable the scale-up fabrication of cell spheroids, and even organoids, offering great potential technology in the biological field.

מחבר זרקור: פיתוח שיטת הרכבה אקוסטית ללא פיגומים לתרבית תאי תלת מימד באיכות גבוהה 

Cell spheroids have been considered one potential model in the field of biological applications. This article describes protocols for scalably generating cell spheroids using a 3D acoustic assembly device, which provides an efficient method for the robust and rapid fabrication of uniform cell spheroids.

התקן הרכבה אקוסטית תלת מימדי לייצור המוני של ספרואידים סלולריים

Cell spheroids are promising three-dimensional (3D) models that have gained wide applications in many biological fields. This protocol presents a method ...