The authors would like to thank Maurice Smith and Marja Talikka for their review of the manuscript.

<ol>
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This study was funded by Philip Morris International.

Here, we have demonstrated the applicability of human organotypic bronchial and nasal tissue models to assess the impact of repeated CS exposure. As an alternative to animal testing, a number of exposure systems were developed for toxicological assessments of aerosol exposure in vitro (e.g., Vitrocell, Cultex, ALICE, etc.). These exposure modules can also be utilized for a toxicological assessment of airborne pollutants, airborne particles, nanoparticles, etc. In this study, we used the Vitrocell system that can accomodate up to 48 different samples simultaneously, allowing for larger scale experiments and lower variability between treatments. For every aerosol exposure in vitro, the risk of tissue culture contamination remains a major risk whose mitigation demands a careful handling of the tissue cultures throughout the experiments.
Measuring particle deposition in real time on the microbalance during the exposure experiment allows to monitor that the CS doses generated from the exposure system are conforming to the expectations. To ensure the accuracy of the measured particle deposition, setting-up the online measurement correctly before the exposure is critial, e.g., setting up the scale to 0. Additionally, because of the difference between the areas of the microbalance (cm2) and the tissue culture insert (0.33 cm2), we adjusted the final calculation to the area of the culture insert: only 33% of the deposition on the microbalance reflect the actual deposition in the culture insert.
Measurement of TEER to ascertain tight-junction barrier functionality and to assess disruption of the epithelial layer is a relatively easy procedure to implement, which we reported here. However, because the bronchial and nasal tissue models contain mucus-producing interspersed goblet cells, apical washing needs to be performed before the TEER measurement. The apical washing is critical because the presence of the mucus layer and the variability of its thickness can bias the measurement of TEER, interferring with the impact of CS exposure. This notion is in agreement with what was reported by Hilgendorf and colleagues, in which the permeability of Caco-2 cells was affected by the co-culturing with the mucus-producing goblet cell line HT29 23. The mucus needs to be washed off prior to TEER measurement, because apical washing right before the exposure may interfere with the tissue responses to CS. Therefore, the measurement is performed three days before exposure, and not right before exposure.
We showed that CYP1A1/CYP1B1 activity could be measured from the organotypic culture models following CS exposure although the activity was only modestly increased by CS. This weak signal can be amplified by a longer incubation of the CYP1A1/1B1 substrate (i.e., luciferin-CEE), for example for 24 hr (data not shown). One of the limitations of the CYP activity measurement in the present work is the absence of normalization to the CYP protein level or to the cell count, which could be taken into consideration for future studies to ensure that the alteration on the enzyme activity is not influenced by the alteration of either the protein level or the cell count.
We reported that CS exposure inhibited the ciliary beating in both the nasal and bronchial tissue models. Similar observations were done in diverse mammalian and non-mammalian models 12. For ciliary beating measurement, ensuring that the tissues are handled and treated in a similar manner is critical, for example if medium change is implemented, it should be applied for all samples. Sutto and colleagues reported that pH affected the mammalian ciliary beating frequency 24. Thus, when comparing beating frequencies between cells treated with different compunds/mixtures, pH adjustment should be considered to minimize the variability of ciliary beating measurement. Moreover, the temperature at which the measurement is conducted is also critical as the frequency of ciliary beating drops with decreasing temperature. To minimize the variability due to these changes, a stage-top incubator, equiped with temperature, CO2, and humidity control was used in this study. Despite this, we observed that the ciliary beating frequencies in the bronchial tissues after CS exposure were highly variable (i.e., increase in one insert and decrease in the other insert), suggesting that the ciliary beating is highly disturbed right after exposure. In contrast, we observed the absence of measurable ciliary beating frequencies in the nasal tissue after CS exposure, suggesting that the response of the nasal tissue is more sensitive and consistent. This is in agreement with a previous publication showing that the nasal tissue has a lower capacity to detoxify as compared with the bronchus 25.
Finally, we showed that gene expression profiling from the organotypic bronchial and nasal tissue models exposed to CS could demonstrate an impact of CS on xenobiotic metabolism. Interestingly, the observed alteration in xenobiotic metabolism in the organotypic bronchial and nasal in vitro models exposed to CS resemble that of the in vivo situation in smokers as discussed in greater detail in a previous publication 15. For gene expression analyses, using the robotic instrument makes a high-throughput analysis possible. Moreover, automatic robotic handling further increases the consistency and accuracy of the gene expression results. Nonetheless, fast collection of the tissue samples was critical to avoid RNA degradation during the RNA extraction. The RNA processing and transcriptomic approaches described here can also be applied to in vivo tissue samples.

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>
		</tr>
		<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>
			<td></td>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Luciferin Detection Reagent&#160;</td>
			<td></td>
			<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.

In results described below, the organotypic tissues were primary human epithelial cells isolated from healthy, non-smoking, Caucasian donors and were reconstituted using fibroblasts 15. 
&#160;
3D bronchial and nasal tissue models 
In vitro bronchial and nasal organotypic models resemble at the cellular level the human respiratory tract epithelium (Figure 2). 
&#160;
CS exposure 
Organotypic bronchial and nasal tissue models can be repeatedly exposed to mainstream CS at the air-liquid interface. The smoke exposure experimental procedure used for the results illustrated here is depicted in Figure 1. 
&#160;
Recording of ciliary beating 
Cilia beating was recorded in the air-exposed tissues as illustrated in the videos. CS exposure was associated with a reduction of the ciliary beating frequency: the stronger peak observed around 4 Hz in the sham exposed and before exposure is not observed any longer after CS exposure (Figure 3). This reduced frequency would make the cilia beating less efficient to remove mucus and infectious agents 22. 
&#160;
Measurement of TEER and CYP1A1/1B1 activity 
TEER was measured 48 hr after the last exposure to ensure the tissue is still functional. CYP1A1/1B1 activity was also measured 48 h after end of exposure as illustrated in Figure 1. The TEER values in the CS-exposed tissues were not significantly different as compared to the air-exposed tissues confirming that there is no major cytotoxic effect of smoke at this concentration. At the 48 h post-exposure, CYP1A1/1B1 activity of the tissues was slightly increased, indicating a modest increase of tissue defense mechanism following the exposure (Figure 4). 
&#160;
Gene expression profiles and network-based systems biology approach to study the impact of CS exposure on the alteration of xenobiotic metabolism 
Tissues were collected at 48 h post-exposure time points. Subsequently, microarray analysis was conducted to generate gene expression profiles of the CS- and air-exposed tissues. Impact of the CS exposure on xenobiotic metabolism was investigated using a network-based systems biology approach leveraged from the gene-expression profiles and as reported earlier 15. Using a network-based systems biology approach shows that CS exposure impacts at the level of backbone nodes in the Xenobiotic Metabolism network model were nicely correlated between the in vitro and in vivo datasets (Figure 5). In contrast, at the level of each individual gene-expression changes, poor correlations were observed between the in vitro (CS-exposed vs. air-exposed) and in vivo (smokers vs. nonsmokers).
<img alt="Figure 1" src="/files/ftp_upload/52325/52325fig1.jpg" /> 
Figure 1. Schematic procedure of the repeated exposure of CS to the organotypic bronchial tissue models. Abbreviations: CYP, cytochrome P450s; CS, cigarette smoke; TEER, transepithelial electrical resistance.
<img alt="Figure 2" src="/files/ftp_upload/52325/52325fig2.jpg" /> 
Figure 2. Organotypic bronchial and nasal epithelial tissue culture models. Cartoon illustrating the bronchial (A) and nasal (B) tissue culture insert at the air-liquid interface. The in vitro models contained ciliated cells shown in the apical layer of the Hematoxylin &#38; Eosin stained cells (C for bronchial, D for nasal) as reported previously 15. The models were co-cultured with fibroblasts that are important for the growth and differentiation of epithelial cells (indicated by arrows). Staining of airway lineage markers: p63 (E for bronchial, F for nasal) and Muc5AC (G for bronchial, H for nasal) are shown as reported previously 15. <a href="https://www.jove.com/files/ftp_upload/52325/52325fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52325/52325fig3.jpg" /> 
Figure 3.&#160;Cilia Beating Frequency. Cilia beating frequency (CBF) was measured in air-exposed and in the culture before and after exposure. Decreased CBF was seen after CS exposure (representative results from two insert are shown as replicate 1 (R1) and replicate 2 (R2)).
<img alt="Figure 4" src="/files/ftp_upload/52325/52325fig4.jpg" /> 
Figure 4. Measurement of CYP1A1/CYP1B1 activity and TEER. Left pannels indicate the CYP1A1/CYP1B1 activity in the bronchial (A) and nasal (B) tissue models. Right panels indicate the TEER measurement in the bronchial (C) and nasal (D) tissue models. Means &#177; SD are shown. Abbreviations: CS, cigarette smoke; CYP, cytochrome P450; TEER, transepithelial electrical resistance; RLU, relative luminescense unit.
<img alt="Figure 5" src="/files/ftp_upload/52325/52325fig5.jpg" /> 
Figure 5. Network-based systems biology approach for the impact assessment of CS exposure in the context of xenobiotic metabolism. The changes of gene expressions levels were used to compute the activation of backbone node of the Xenobiotic Metabolism network model (A) as reported in a previous publication 15. The figure on the right illustrates the concept of the quantification of the backbone nodes, also known as &#8220;differential network backbone value,&#8221; using the NPA approach. The blue ovals represent the activity of the backbone nodes (i.e., the functional layer) and the green balls represent the expression of genes (i.e., the transcriptional layer). Impacts of CS exposure in organotypic bronchial tissues (in vitro, CS-exposed vs. air-exposed) at the 48 h of postexposure were compared to those of smoking on human bronchial epithelial collected by bronchoscopy and nasal epithelial by burshing the inferior turbinate (GSE16008) (in vivo, smokers vs. nonsmokers) (B). Insets, correlation of the gene expression changes between the in vitro and in vivo datasets. Abbreviations: CS, cigarette smoke; NPA, network perturbation amplitude. <a href="https://www.jove.com/files/ftp_upload/52325/52325fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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.

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 ...

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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.

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.

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.

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 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.

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.

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.

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.

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

Scalable Generation of Uniform Cell Spheroids Using a 3D Acoustic Assembly Device

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.

Author Spotlight: Development of a Scaffold-Free Acoustic Assembly Method for High-Quality 3D Cell Spheroid Culture 

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.

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

Comparative Study of Decellularization Techniques for Bovine Lung ECM Hydrogels

Development and Characterization of Decellularized Lung Extracellular Matrix Hydrogels

The use of extracellular matrix (ECM)-derived hydrogels in tissue engineering has become increasingly popular, as they can mimic cells&#39; natural environment in vitro. However, maintaining the native biochemical content of the ECM, achieving mechanical stability, and comprehending the impact of the decellularization process on the mechanical properties of the ECM hydrogels are challenging. Here, a pipeline for decellularization of bovine lung tissue using two different protocols, downstream characterization of the effectiveness of decellularization, fabrication of reconstituted decellularized lung ECM hydrogels and assessment of their mechanical and cytocompatibility properties were described. Decellularization of the bovine lung was pursued using a physical (freeze-thaw cycles) or chemical (detergent-based) method. Hematoxylin and Eosin staining was performed to validate the decellularization and retention of major ECM components. For the evaluation of residual collagen and sulfated glycosaminoglycan (sGAG) content within the decellularized samples, Sirius red and Alcian blue staining techniques were employed, respectively. Mechanical properties of the decellularized lung ECM hydrogels were characterized by oscillatory rheology. The results suggest that decellularized bovine lung hydrogels can provide a reliable organotypic<span style="font-size: 11pt; font-family: Calibri, sans-serif; color: black; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&#160;alternative to commercial ECM products by retaining most native ECM components. Furthermore, these findings reveal that the decellularization method of choice significantly affects gelation kinetics as well as the stiffness and viscoelastic properties of resulting hydrogels.

Author Spotlight: Optimizing Bovine Lung Decellularization for Organotypic Hydrogels

The protocol elucidates two distinct decellularization methodologies applied to native bovine pulmonary tissues, providing a comprehensive account of their respective characterizations.

The use of extracellular matrix (ECM)-derived hydrogels in tissue engineering has become increasingly popular, as they can mimic cells&#39; natural ...