저자는 원고의 자신의 검토를 위해 모리스 스미스와 Marja Talikka에게 감사의 말씀을 전합니다.

<ol>
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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+biological+impact+using+transcriptomic+data+and+mechanistic+network+models.">Quantitative assessment of biological impact using transcriptomic data and mechanistic network models.</a> Toxicology and applied pharmacolog. 272, 863-878 (2013).</li><li>Teff, Z., Priel, Z., Gheber, L. 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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이 연구는 필립 모리스 인터내셔널 (Philip Morris International)에 의해 투자되었다.

여기서는 반복 CS 노출의 영향을 평가하는 인간의 Organotypic 기관지 비강 조직 모델의 적용 가능성을 보여 주었다. 동물 실험에 대한 대안으로, 노광 시스템의 수는 (예., Vitrocell, Cultex, ALICE 등) 시험관 내에서 에어로졸 노출 독성 평가를 위해 개발되었다. 이러한 노광 모듈도 본 연구 등 공수 오염 물질, 공기 중의 입자, 나노 입자의 독성 학적 평가를 위해 이용 될 수 있고, 우리는 더 큰 규모의 실험 간의 낮은 가변성을 허용 동시에 48 가지의 다른 샘플들까지 수용 할 수 Vitrocell 시스템을 사용 치료. 시험관 내에서 모든 에어로졸 노출, 조직 배양 오염의 위험은 저감 그 실험에 걸쳐 조직 배양의 취급에주의를 요구하는 주요 위험 남아있다. E 중 미량 천칭에 실시간 입자 침착을 측정xposure 실험은 노출 시스템에서 생성 된 CS 량은 기대에 부합하는 것을 모니터링 할 수 있습니다. 설정해서 온라인 측정을 정확하게 노광 전에 것은, 예 critial 때문에 마이크로 저울 (cm 2)의 영역의 차이, 또한 0으로 스케일을 설정하고, 측정 된 입자의 증착의 정확성 조직 배양 인서트 (0.33 cm 2), 우리는 배양 인서트의 영역에 최종 계산을 조정 된 미량 천칭에 증착 단지 33 %가 배양 인서트의 실제 증착을 반영한다. 꽉 접합 장벽 기능을 확인하고 상피 층의 중단을 평가하기 위해 봉사자의 측정은 우리가 여기에보고 구현하기 비교적 쉬운 과정이다. 비강 및 기관지 조직 모델 점액 생산 산재 배 세포를 포함하기 때문에, 정점 세척 봉사자 MEAS 전에 행할 필요하여 연산. 정점 세척 중요하므로 점액층의 존재 및 CS 노출의 영향 interferring 두께 캔 바이어스 티에르의 측정 가변성. 이 개념은 Hilgendorf 및 카코-2 세포의 투과성이 점액을 생산하는 술잔 세포 라인 HT29 (23)와 공동 배양에 의해 영향을 받았다있는 동료들에 의해보고 된 것과 일치한다. 바로 노출 전에 혀끝의 세척 CS에 대한 조직 반응을 방해 할 수 있기 때문에 점액 전에 봉사자 측정에 씻어해야합니다. 따라서, 측정은 노출 3 일 전에 수행 및 노출되기 직전되지 않습니다. 우리는 활동이 완만 한 CS 증가되었지만 CYP1A1 / CYP1B1 활동이 CS 노출 다음의 Organotypic 문화 모델에서 측정 될 수 있음을 보여 주었다. 이 약한 신호는 CYP1A1 / 1B1 기판의 장기간 배양에 의해 증폭 될 수있다 (예., 루시페린 - CEE), 예컨대 대24 시간 (데이터 미기재). 본 연구에서 CYP 활성 측정의 한계 중 하나는 효소 활성에 변화가 영향을받지 않도록 CYP 단백질 수준 또는 미래 연구를 위해 고려 될 수있는 세포 수로 정규화 결석 단백질 수준 또는 세포 수의 변화 중 하나에 의하여. 우리는 CS 노출은 비강과 기관지 조직 모델을 모두 제치고 섬모를 억제 보도했다. 비슷한 관찰 다양한 포유 동물 및 비 - 포유 동물 모델 (12)에서 수행 하였다. 섬모가 측정 치는 조직을 유사한 방식으로 처리하고 처리하도록 할 변화 매체가 구현되는 경우, 모든 샘플에 적용되어야한다, 예를 들면, 중요하다. Sutto와 동료들은 pH가 주파수 (24)를 때리고 포유 동물의 섬모에 영향을 보도했다. 따라서, 다른 compunds / 혼합물의 pH로 처리 한 세포 사이의 주파수를 치고 비교할 때조정은 모양체 박동 측정의 변동을 최소화하기 위해 고려되어야한다. 모양체 고해 주파수가 감소함에 따라 온도 강하 더욱이, 측정이 수행되는 온도는 중요하다. 본 연구에서 사용 된 온도, CO 2, 조습 갖추고 이러한 변화, 탑 스테이지 배양기에 의한 변동을 최소화. 그럼에도 불구하고, 우리는 CS 노출 후 기관지 조직에 주파수를 치는 섬모는 섬모 구타 노출 후 매우 방해 권리가 있음을 시사 매우 변수 (예., 하나 삽입 증가하고 다른 삽입 감소)이라고 관찰했다. 대조적으로, 우리는 비강 조직의 반응이 더 민감하고 일관성이 있음을 시사 CS 노광 후의 비강 조직에서 측정 모양체 비팅 주파수의 유무를 관찰했다. 이 비강 조직으로 독을 제거하기 위해 낮은 용량을 가지고 있음을 보여주는 이전의 출판물과 일치한다기관지 (25)와 비교. 마지막으로, 우리는 CS에 노출의 Organotypic의 기관지와 코 조직 모델에서 프로파일 유전자 발현이 생체 이물 대사에 CS의 영향을 설명 할 수 있음을 보여 주었다. 이전 출판 (15)에 자세히 설명한 바와 같이 흥미롭게도, CS에 노출 체외 모델에서 관찰의 Organotypic의 기관지에서 생체 이물 대사 변경과 코는 흡연자의 생체 내 상황의 유사. 유전자 발현 분석을 위해, 로봇 기기를 사용하면 높은 처리량 분석을 가능하게한다. 또한, 자동 핸들링 로봇은 상기 유전자 발현 결과의 일관성 및 정확성을 증가시킨다. 그럼에도 불구하고, 조직 샘플의 신속한 수집 RNA 추출 중에 RNA의 분해를 방지하는 것이 중요했다. RNA 프로세싱 및 여기서 설명 transcriptomic 접근법은 또한 생체 조직 샘플에 적용될 수있다.

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>
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		<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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		<tr height="21">
			<td height="21" style="height:21px;">3R4F reference cigarette</td>
			<td>University of Kentucky</td>
			<td>http://www2.ca.uky.edu/refcig/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">30-port carousel smoking machine SM2000</td>
			<td>Philip Morris, Int.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CiliaMetrix camera and software</td>
			<td>La Haute &#201;cole de Gestion (HESGE), Geneva, Switzlerland</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica DMIL microscope&#160;</td>
			<td>Leica, Heerbrugg, Switzerland</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LED light source</td>
			<td>Titan Tool Supplies, Buffalo, NY</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Chopstick Electrode STX-2</td>
			<td>World Precision Instruments</td>
			<td>http://www.wpiinc.com/products/physiology/stx2-chopstick-electrode-set-for-evom2/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EVOMXTM Epithelial Voltohmmeter&#160;</td>
			<td>World Precision Instruments</td>
			<td>http://www.wpiinc.com/products/physiology/evom2-evom2-epithelial-voltohmmeter-for-teer/</td>
			<td></td>
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			<td height="21" style="height:21px;">Luciferin Detection Reagent&#160;</td>
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			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MagNA Lyser Instrument&#160;</td>
			<td>Roche</td>
			<td>http://www.roche.com/products/product-details.htm?region=us&#38;type=product&#38;id=66</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">chloroform&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sial/288306?lang=en&#38;region=</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAcube&#160;</td>
			<td>Qiagen</td>
			<td align="right">9001882</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NanoDrop</td>
			<td>Thermo Scientific</td>
			<td>http://www.nanodrop.com/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agilent 2100 Bioanalyzer&#160;</td>
			<td>Agilent</td>
			<td>http://www.genomics.agilent.com/en/Bioanalyzer-System/2100-Bioanalyzer-Instruments/?cid=AG-PT-106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Affymetrix GeneChip High throughput 3&#8217;IVT Express Kit</td>
			<td>Affymetrix</td>
			<td>http://www.affymetrix.com/catalog/prod370001/AFFY/High-Throughput-(HT)-Whole-Transcript-(WT)-Kit</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scanner 3000 7G&#160;</td>
			<td>Affymetrix</td>
			<td>http://www.affymetrix.com/catalog/131503/AFFY/Scanner-3000-7G</td>
			<td></td>
		</tr>
	</tbody>
</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.

아래에 설명 된 결과에서의 Organotypic 조직 건강, 금연로부터 격리 차 인간 상피 세포, 백인 기증자이었고, 섬유 아세포 (15)를 사용하여 재구성 하였다. 3D 기관지와 코 조직 모델 체외 기관지와 코의 Organotypic 모델에서 세포 수준 인간의 호흡기 상피 세포 (그림 2)에서 유사. CS 노출 비강의 Organotypic 기관지 조직 모델 반복적 공기 - 액체 계면에서 주류 CS에 노출 될 수있다. 여기에 도시 된 결과를 사용 흡연 노출 실험 절차는도 1에 도시되어있다. 섬모 박동의 기록 속눈썹 박동이 동영상에 도시 된 바와 같이 공기에 노출 된 조직에서 기록되었다. CS 노출은 섬모 박동 주파수의 감소와 관련이 : 주위에 관찰 강한 피크를노출 노출 된 가짜 및 이전 4 Hz로는 CS 노출 (그림 3) 이후 더 이상 관찰되지 않는다. 이 감소 된 주파수는 점액과 감염원 (22)를 제거 덜 효율적 치는 속눈썹을 만들 것입니다. 봉사자 및 CYP1A1 / 1B1 활동의 측정 봉사자는 조직이 여전히 기능에 마지막 노출 후 48 시간을 측정 하였다. 도 1에 도시 된 바와 같이, CYP1A1 / 1B1 활성은 노출 종료 후 48 시간을 측정 하였다. CS에 노출 된 조직에서 티에르 값이 크게 차이가 없었다 연기의 더 큰 세포 독성 효과가 있다는 것을 확인하는 공기에 노출 된 조직에 비해 이 농도. 48 시간 후 - 노출에서, 조직의 CYP1A1 / 1B1 활동은 약간 노출 (그림 4) 다음과 같은 조직의 방어 메커니즘의 완만 한 증가를 나타내는 증가 하였다. 유전자 발현 양상 및 네트워크 기반 시스템 양방향ology 접근법 생체이 대사의 변화에​​ CS 노출의 영향을 연구 조직을 48 시간 노출 후 시점에서 수집 하였다. 이어서, 마이크로 어레이 분석은 유전자 발현 프로필 및 CS- 공기에 노출 된 조직을 생성하기 위해 수행 하였다. 생체 이물 대사에 CS 노출의 영향은 유전자 발현 양상에서 활용 네트워크 기반 시스템 생물학 접근 방식을 사용하여 조사하고로 이전 (15) 보도했다. 네트워크 기반 시스템 생물학 접근 방식을 사용하여 생체 이물 대사 네트워크 모델의 백본 노드의 수준에서 CS 노출에 영향을 멋지게 (그림 5) 시험 관내 사이 및 생체 데이터 세트의 상관 관계를 보여줍니다. 대조적으로, 각각의 유전자 발현 변화의 차원에서, 열악한 상관 관계 관내 (대 비 흡연자가 흡연) 및 생체 내 (공기에 노출 된 노출 대 CS) 사이에서 관찰되었다. 의 Organotypic 기관지 조직 모델 CS의 반복 노출의 그림 1. 도식 절차 약어 :. CYP, 사이토 크롬 P450s; CS, 담배 연기, 봉사자, transepithelial 전기 저항. <img alt="그림 2" src="/files/ftp_upload/52325/52325fig2.jpg" /> 도 2의 Organotypic 비강 기관지 상피 조직 배양 모델. 만화 공기 - 액체 계면에서 기관지 (A)와 코 (B) 조직 배양 삽입물을 도시. 이전 15보고 체외 모델은 헤 마톡 실린 및 에오신 염색 된 세포의 꼭대기 층에 표시된 섬 모세포 (기관지에 대한 C, 비강 용 D)를 포함. 모델은 중요 아세포와 공동 배양 하였다<html> 성장 및 상피 세포의 분화를 위해 (화살표로 표시). 기도 혈통 마커의 얼룩 : 이전에 15보고 P63 (코 기관지에 대한 E, F)와 MUC5AC (기관지에 대한 G는 코에 대한 H)가 표시됩니다. <a href="https://www.jove.com/files/ftp_upload/52325/52325fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 3" src="/files/ftp_upload/52325/52325fig3.jpg" /> 그림 3. 섬모 치는 주파수. 섬모 박동 주파수 (CBF)는 전 및 노출 후 공기에 노출과 문화 측정 하였다. CBF는 CS 노출 후 본 낮게 나타났다 (두 삽입 대표 결과가 복제 1 (R1로 표시) 및 복제되는 2 (R2)). 광고 / 52325 / 52325fig4.jpg &quot;/&gt; 그림 4. CYP1A1 / CYP1B1 활동과 봉사자의 측정. 왼쪽 pannels는 기관지 (A)과 코 (B) 조직 모델에서 CYP1A1 / CYP1B1 활동을 나타냅니다. 오른쪽 패널은 기관지 (C)과 코 (D) 조직 모델 티에르 측정을 나타낸다. SD ± 수단이 표시됩니다. 요약 : CS, 담배 연기, CYP 시토크롬 P450; 봉사자, transepithelial 전기 저항; RLU, 상대 luminescense 장치. <img alt="그림 5" src="/files/ftp_upload/52325/52325fig5.jpg" /> 생체이 대사의 맥락에서 CS 노출의 영향 평가 그림 5. 네트워크 기반 시스템 생물학 방법. 유전자 발현 수준의 변화는 생체이 대사 네트워크 모델의 백본 노드의 활성화 (A)를 계산하는 데 사용 된 </<html> 강한&gt; 이전 게시 (15)에보고. 오른쪽 그림은 NPA 방식을 사용하여 &quot;차동 네트워크 백본 값」라고도 백본 노드의 정량의 개념을 도시한다. 청색 타원은 백본 노드의 활성을 나타낸다 (예., 기능 층) 및 녹색 공은 유전자의 발현을 나타낸다 (예., 전사 층). 의 Organotypic 기관지 조직에서 CS 노출의 영향 GSE16008 (노출 후의 48 시간에서 하 비갑개를 burshing에 의해 기관지와 코 점막 상피에 의해 수집 된 인간 기관지 상피에 흡연의 것과 비교 하였다 (체외은 공기에 노출이 대 CS는 노출) ) (생체, 흡연자 대 비 흡연자의) (B). 인 세트는, 유전자 발현의 상관 관계는 시험 관내 및 생체 내 사이에 데이터 셋 변경한다. 요약 : CS, 담배 연기, NPA 네트워크 교란 진폭.ove.com/files/ftp_upload/52325/52325fig5large.jpg &quot;대상 =&quot;_ 빈 &quot;&gt;이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.

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.

전체 담배 연기에의 Organotypic 3D 기관지와 코 조직 문화 모델의 반복 노출의 영향 평가

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 조회수.  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.

비디오: 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.

메인 스트림 담배 연기에 자유 래디 칼을 감지하고 가스 단계 청소를위한 프로토콜

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.  ...

연구

생체공학

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.

인간의 3 차원의 조직 공학<em&gt; 시험관</em&gt; 종양 테스트 시스템

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.

3 차원 세포 배양 모델의 질량 분석 이미징을위한 준비 전략 샘플

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.

연골과 피부 조직 아날로그 소설 수동 Bioink Precellularization에 대 한 단위 기술 혼합을 이용 하 여 Bioprinting

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.

전통적인 담배 연기와 전자 담배 연 무질에서 액체 추출의 세대에 대 한 장치를 운영 하는 마이크로컨트롤러

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

Preclinical Cardiac Electrophysiology Assessment by Dual Voltage and Calcium Optical Mapping of Human Organotypic Cardiac Slices

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between animal and clinical trials. Numerous animal and cell models have been used to examine the effects of drugs, yet these responses often differ in humans. Human cardiac slices offer an advantage for drug testing in that they are directly derived from viable human hearts. In addition to having preserved multicellular structures, cell-cell coupling, and extracellular matrix environments, human cardiac tissue slices can be used to directly test the effect of innumerable drugs on adult human cardiac physiology. What distinguishes this model from other heart preparations, such as whole hearts or wedges, is that slices can be subjected to longer-term culture. As such, cardiac slices allow for studying the acute as well as chronic effects of drugs. Furthermore, the ability to collect several hundred to a thousand slices from a single heart makes this a high-throughput model to test several drugs at varying concentrations and combinations with other drugs at the same time. Slices can be prepared from any given region of the heart. In this protocol, we describe the preparation of left ventricular slices by isolating tissue cubes from the left ventricular free wall and sectioning them into slices using a high precision vibrating microtome. These slices can then either be subjected to acute experiments to measure baseline cardiac electrophysiological function or cultured for chronic drug studies. This protocol also describes dual optical mapping of cardiac slices for simultaneous recordings of transmembrane potentials and intracellular calcium dynamics to determine the effects of the drugs being investigated.

This protocol describes the procedure for sectioning and culturing human cardiac slices for preclinical drug testing and details the use of optical mapping for recording transmembrane voltage and intracellular calcium signals simultaneously from these slices.

인간 조직형 심장 슬라이스의 이중 전압 및 칼슘 광학 매핑에 의한 전임상 심장 전극생리학 평가

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between ...

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.

장기 나노 물질 노출 다음 체외 물질 독성 테스트를 위한 고급 3D 간 모델

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.

하이드로겔 기반 3D 문화 모델을 자동화및 표준화방식으로 제조하는 오픈 소스 기술 플랫폼

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.

Y-27632를 사용하여 1차 인간 칭기발 상피 세포의 격리 및 배양

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

3D Acoustic Assembly Device를 사용한 균일한 세포 스페로이드의 확장 가능한 생성

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.

저자 스포트라이트: 고품질 3D 세포 스페로이드 배양을 위한 스캐폴드가 없는 음향 조립 방법 개발 

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.

세포 스페로이드의 대량 생산을 위한 3차원 음향 조립 장치

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