Name: a combined 3d tissue engineered in vitro/in silico lung tumor model for predicting drug effectiveness in specific mutational backgrounds
Uploaded: 2016-04-06T14:00:00
Description: Watch this Scientific Journal Video about A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds at JoVE.com

This research was sponsored by the Center for Interdisciplinary Clinical Research (IZKF, grant BD247) of the University Hospital of Wuerzburg and the Bayern Fit program (granted to Heike Walles).

1. Two-dimensional (2D) Cell Culture
<ol>
	<li>Commercially obtain tumor cell line HCC827 (DSMZ). Culture the lung adenocarcinoma cell line HCC827 (EGFR mutated, KRAS wild-type) in RPMI-1640 supplemented with 20% FCS. Change medium every 2 - 3 days. Split the cells twice a week. Cells are used until passage 20 is reached.</li>
	<li>Commercially obtain tumor cell line A549 (DSMZ). Culture the lung carcinoma cell line A549 (EGFR wild-type, KRAS mutated) in RPMI-1640 supplemented with 1...

<ol>
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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gefitinib+or+carboplatin-paclitaxel+in+pulmonary+adenocarcinoma.">Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma.</a> N Engl J Med. 361, 947-957 (2009).</li><li>Maemondo, 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=Gefitinib+or+chemotherapy+for+non-small-cell+lung+cancer+with+mutated+EGFR.">Gefitinib or chemotherapy for non-small-cell lung cancer with mutated EGFR.</a> N Engl J Med. 362, 2380-2388 (2010).</li><li>Rosell, R., 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=Erlotinib+versus+standard+chemotherapy+as+first-line+treatment+for+European+patients+with+advanced+EGFR+mutation-positive+non-small-cell+lung+cancer+(EURTAC):+a+multicentre,+open-label,+randomised+phase+3+trial.">Erlotinib versus standard chemotherapy as first-line treatment for European patients with advanced EGFR mutation-positive non-small-cell lung cancer (EURTAC): a multicentre, open-label, randomised phase 3 trial.</a> Lancet Oncol. 13, 239-246 (2012).</li><li>Sequist, L. 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We have established a combined in vitro/in silico tumor test system for biomarker-guided treatment predictions. The in vitro model evaluates different important aspects of compound actions such as changes of tumor cell proliferation and apoptosis on a specific mutational background that can also be simulated in silico17. Here, we present the standardized protocol for 3D tumor model generation and compound testing including quantification of proliferation and apoptosis and the establi...

The pharmaceutical industry is facing high attrition rates of up to 95% in the field of cancer treatment in the clinical phase causing enormous costs 1-5. One reason for this deficit is the fact that currently efficacy of potential new compounds is assessed in large-scale screenings on 2D cell cultures of cancer cell lines or in animal models. Animal models have a higher complexity but there are crucial differences between mice and men 6,7. In the last decade, 3D cancer models using different approaches have been generated to bridge the gap between 2D culture of cancer cell lines and a complex in vivo tumor 6,8,9. The impact of 3D environment on cell differentiation and also on signaling has been shown in several studies years ago (e.g., by Mina Bissell) 10,11. Today, many 3D cell culture models are available such as spheroid cultures, hydrogels or microfluidic chips 12-16. Even though these models enhance complexity compared to conventional 2D culture systems, they mostly lack a tissue microenvironment that is known to have tumor-supporting effects and also impacts drug efficacy.
To address this issue, we generated a 3D tumor model based on a biological scaffold called SISmuc (small-intestine-submucosa + mucosa) that is derived from a decellularized porcine jejunum. Thereby, the tissue architecture and important components of the ECM such as different collagens as well as the basement membrane structure are preserved 17. This unique feature is crucial for tumor model generation of carcinomas that arise from epithelia and comprise about 80% of solid tumors. Furthermore, the proliferation rate in our tissue-engineered tumor model is reduced compared to the artificially high rates achieved in 2D culture. As proliferation is an important parameter in assessing drug efficacy, drug testing is enabled in our model in more similar conditions to in vivo tumors 17.
In order to evaluate the potential of our model to predict biomarker-dependent drug efficacy correctly, we here present data for two different lung cancer cell lines that differ in their EGFR-biomarker status. This mutational status has started to be determined routinely in NSCLC patients. Targeted treatments with TKIs such as the EGFR-inhibitor gefitinib against tumors bearing an activating EGFR mutation show superior outcomes compared to those with platinum-based chemotherapy 18-21.
We established several read-out techniques that are relevant for evaluating compound efficacy. Furthermore, after TGF-beta-1 stimulation we are able to investigate compound actions in tumor cells that started the EMT process, which is thought to be an important step in malignant transformation 22,23 and which is connected to drug resistance 24.
The 3D tumor model allow monitoring cell-specific responses to targeted treatments, chemotherapy, or drug combinations with good contrasts. To further enhance and speed up drug-screening and to encounter resistance, this is complemented by an in silico simulation. Based on a few experiments, the tumor response can be predicted in silico regarding the outcome for a full range of drugs and their combinations.

<table border="0" cellpadding="0" cellspacing="0" width="808">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Bioreactors</td>
			<td style="width:261px;">Chair of Tissue Engineering and Regenerative Medicine, W&#252;rzburg (GER)</td>
			<td style="width:119px;"></td>
			<td style="width:212px;">Bioreactor setup</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">BioVoxxel Toolbox (ImageJ / Fiji)</td>
			<td style="width:261px;">Jan Brocher, Thorsten Wagner, https://github.com/biovoxxel/BioVoxxel_Toolbox</td>
			<td style="width:119px;"></td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Cell crowns</td>
			<td style="width:261px;">Chair of Tissue Engineering and Regenerative Medicine, W&#252;rzburg (GER)</td>
			<td style="width:119px;"></td>
			<td style="width:212px;">for static 3D culture</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CellDesigner</td>
			<td style="width:261px;">http://www.celldesigner.org/</td>
			<td style="width:119px;">-</td>
			<td style="width:212px;">This software was used for drawing the network.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">citrate buffer stock solution (10x)</td>
			<td style="width:261px;">in house production</td>
			<td style="width:119px;"></td>
			<td style="width:212px;">42 g/l Citric acid monohydrate, 17.,6 g/l Sodium hydroxide pellets in deionized water, pH 6,.0, stored at RT.&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">citrate buffer working solution</td>
			<td style="width:261px;">in house production</td>
			<td style="width:119px;"></td>
			<td style="width:212px;">10 % Citrate buffer stock solution in demineralized water, stored at RT.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Citric acid monohydrate</td>
			<td style="width:261px;">VWR, Darmstadt (GER)</td>
			<td style="width:119px;">1002441000</td>
			<td style="width:212px;">used for the citrate buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cover slips</td>
			<td style="width:261px;">VWR, Darmstadt (GER)</td>
			<td>631-1339</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">DAPI Fluoromount-GTM</td>
			<td style="width:261px;">SouthernBiotech, Birmingham (USA)</td>
			<td style="width:119px;">SBA-0100-20</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Databases such as KEGG, HPRD and QIAGEN (Genes &#38; Pathways)</td>
			<td style="width:261px;">http://www.genome.jp/kegg/pathway.html; http://www.hprd.org/; https://www.qiagen.com/de/geneglobe/</td>
			<td style="width:119px;">-</td>
			<td style="width:212px;">Different known literature databases were used for generating the network topology.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Female Luer Lug Style Tee</td>
			<td style="width:261px;">Mednet, M&#252;nster (GER)</td>
			<td style="width:119px;">FTLT-1</td>
			<td style="width:212px;">Bioreactor setup</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Female Luer Thread Style with 5/16&#34; Hex to 1/4-28 UNF Thread</td>
			<td style="width:261px;">Mednet, M&#252;nster (GER)</td>
			<td style="width:119px;">SFTLL-J1A&#160;</td>
			<td style="width:212px;">Bioreactor setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal calf serum</td>
			<td style="width:261px;">Bio&#38;SELL, Feucht (GER)</td>
			<td style="width:119px;">FCS.ADD.0500</td>
			<td style="width:212px;">not heat-inactivated</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Gefitinib</td>
			<td style="width:261px;">Absource Diagnostics GmbH, M&#252;nchen (GER)</td>
			<td style="width:119px;">S1025-100 mg</td>
			<td style="width:212px;">100 mM stock solution with DMSO</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Glas flask (Schott, GER) provided with glas hose connection</td>
			<td style="width:261px;">Weckert, Kitzingen (GER)</td>
			<td style="width:119px;">custom made</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Histofix 4 % (Paraformaldehyd)</td>
			<td style="width:261px;">Carl Roth, Karlsruhe (GER)</td>
			<td style="width:119px;">P087.1</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hose coupling</td>
			<td style="width:261px;">Mednet, M&#252;nster (GER)</td>
			<td>CC-9</td>
			<td style="width:212px;">Bioreactor setup</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Incubator for bioreactors</td>
			<td style="width:261px;">Chair of Tissue Engineering and Regenerative Medicine, W&#252;rzburg (GER)</td>
			<td style="width:119px;"></td>
			<td style="width:212px;">Bioreactor setup</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">M30 CytoDeathTM ELISA</td>
			<td style="width:261px;">Peviva, Bromma (SWE)</td>
			<td style="width:119px;">10900</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Male Luer Integral Lock Ring</td>
			<td style="width:261px;">Mednet, M&#252;nster (GER)</td>
			<td style="width:119px;">MTLL230-J1A</td>
			<td style="width:212px;">Bioreactor setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Moisture chamber</td>
			<td style="width:261px;">custom made</td>
			<td style="width:119px;"></td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Mouse anti Pan-Cytokeratin</td>
			<td style="width:261px;">Sigma-Aldrich, Munich (GER)&#160;&#160;</td>
			<td style="width:119px;">C2562-2ML</td>
			<td style="width:212px;">Clone C-11+PCK-26+CY-90+KS-1A3+M20+A53-B/A2, used 1/100 for immunofluorescence</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Needlefree Swabable Valve Female Luer</td>
			<td style="width:261px;">Mednet, M&#252;nster (GER)</td>
			<td>NVFMLLPC</td>
			<td style="width:212px;">Bioreactor setup, for sampling, gamma-sterilized</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">O-Ring MVQ 10 red 37*3 mm</td>
			<td style="width:261px;">Arcus Dichtelemente, Seevetal (GER)</td>
			<td style="width:119px;">21444</td>
			<td>O-ring large, Bioreactor setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">O-Ring MVQ 70 red 27*2.5 mm</td>
			<td style="width:261px;">Arcus Dichtelemente, Seevetal (GER)</td>
			<td style="width:119px;">19170</td>
			<td>O-ring small, Bioreactor setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PAP pen</td>
			<td style="width:261px;">Dako, Hamburg (GER)</td>
			<td>S002</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Paraffin</td>
			<td style="width:261px;">Carl Roth, Karlsruhe (GER)</td>
			<td style="width:119px;">6642.6</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Peristaltic pump</td>
			<td style="width:261px;">Ismatec, Wertheim-Mondfeld (GER)</td>
			<td style="width:119px;"></td>
			<td style="width:212px;">Bioreactor setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phosphate Buffered Saline</td>
			<td style="width:261px;">Sigma-Aldrich, Munich (GER)&#160;&#160;</td>
			<td style="width:119px;">D8537-6x500ml</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pump tubing cassette</td>
			<td style="width:261px;">Ismatec, Wertheim (GER)</td>
			<td>IS 3710</td>
			<td style="width:212px;">Bioreactor setup</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Rabbit anti Ki67</td>
			<td style="width:261px;">Abcam, Cambridge (UK)</td>
			<td style="width:119px;">ab16667</td>
			<td style="width:212px;">Clone SP6, used for 1/100 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rabbit anti Vimentin</td>
			<td style="width:261px;">Abcam, Cambridge (UK)</td>
			<td style="width:119px;">ab92547</td>
			<td style="width:212px;">used 1/100 for IF</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">RPMI-1640 medium</td>
			<td style="width:261px;">Life technologies, Darmstadt (GER)</td>
			<td style="width:119px;">61870-044</td>
			<td style="width:212px;">warm in 37&#176;C waterbath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Silicone tube</td>
			<td style="width:261px;">Carl Roth GmbH, Karlsruhe (GER)</td>
			<td style="width:119px;">HC66.1</td>
			<td style="width:212px;">Bioreactor setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Hydroxide</td>
			<td style="width:261px;">Sigma-Aldrich, M&#252;nchen (GER)</td>
			<td>30620-1KG-R</td>
			<td style="width:212px;">used for the citrate buffer</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">SQUAD</td>
			<td style="width:261px;">http://sbos.eu/docu/docu/SQUAD/doku.php.htm</td>
			<td style="width:119px;">-</td>
			<td style="width:212px;">This software was used for performing the semiquantitative simulations.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sterile air filter, pore size 0.2 &#181;m</td>
			<td style="width:261px;">Sartorius Stedium Biotech, G&#246;ttlingen (GER)</td>
			<td style="width:119px;">16596-HYK</td>
			<td style="width:212px;">Bioreactor setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe Luer Lok 5ml</td>
			<td>BD Biosciences, Heidelberg (GER)</td>
			<td>309649</td>
			<td style="width:212px;">for bioreactor sampling</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tissue culture test plates: 6-,&#160;&#160;&#160;&#160;&#160; 12-, 24-, 96- well</td>
			<td style="width:261px;">TPP Techno Plastic Products AG, Trasadingen (GER)</td>
			<td style="width:119px;">92006, 92012, 92024, 92048&#160;</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Transforming growth factor-beta 1 (TGF-&#946;1) with carrier</td>
			<td style="width:261px;">Cell Signaling, Frankfurt (GER)</td>
			<td style="width:119px;">8915LC</td>
			<td style="width:212px;">stock solution in sterile citrate buffer pH 3.0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X-100</td>
			<td style="width:261px;">Sigma-Aldrich, M&#252;nchen (GER)</td>
			<td style="width:119px;">X100-1L</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tween-20</td>
			<td style="width:261px;">Sigma-Aldrich, M&#252;nchen (GER)</td>
			<td style="width:119px;">P7949-500ml</td>
			<td style="width:212px;">for washing buffer of immunofluorescent staining</td>
		</tr>
	</tbody>
</table>

a combined 3d tissue engineered in vitro/in silico lung tumor model for predicting drug effectiveness in specific mutational backgrounds

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific mutational background. The model is generated on a decellularized porcine scaffold that reproduces tissue-specific characteristics regarding extracellular matrix composition and architecture including the basement membrane. We standardized a protocol that allows artificial tumor tissue generation within 14 days including three days of drug treatment. Our article provides several detailed descriptions of 3D read-out screening techniques like the determination of the proliferation index Ki67 staining&#39;s, apoptosis from supernatants by M30-ELISA and assessment of epithelial to mesenchymal transition (EMT), which are helpful tools for evaluating the effectiveness of therapeutic compounds. We could show compared to 2D culture a reduction of proliferation in our 3D tumor model that is related to the clinical situation. Despite of this lower proliferation, the model predicted EGFR-targeted drug responses correctly according to the biomarker status as shown by comparison of the lung carcinoma cell lines HCC827 (EGFR -mutated, KRAS wild-type) and A549 (EGFR wild-type, KRAS-mutated) treated with the tyrosine-kinase inhibitor (TKI) gefitinib. To investigate drug responses of more advanced tumor cells, we induced EMT by long-term treatment with TGF-beta-1 as assessed by vimentin/pan-cytokeratin immunofluorescence staining. A flow-bioreactor was employed to adjust culture to physiological conditions, which improved tissue generation. Furthermore, we show the integration of drug responses upon gefitinib treatment or TGF-beta-1 stimulation - apoptosis, proliferation index and EMT - into a Boolean in silico model. Additionally, we explain how drug responses of tumor cells with a specific mutational background and counterstrategies against resistance can be predicted. We are confident that our 3D in vitro approach especially with its in silico expansion provides an additional value for preclinical drug testing in more realistic conditions than in 2D cell culture.

On the basis of the SISmuc scaffold (Figure 2A to C), we established a standardized operating protocol for the generation, stimulation and treatment of a 3D tumor test system (Figure 2D). This model enables the determination of the proliferation index and the quantification of apoptosis using M30-ELISA as shown in Figure 1 and Figure 3, respectively. Figure 3 shows representative H&#38;E staining of A549 ...

Watch this Scientific Journal Video about A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds at JoVE.com

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

We present a three-dimensional (3D) lung cancer model based on a biological collagen scaffold to study sensitivity towards non-small-cell-lung-cancer-(NSCLC)-targeted therapies. We demonstrate different read-out techniques to determine the proliferation index, apoptosis and epithelial-mesenchymal transition (EMT) status. Collected data are integrated into an in silico model for prediction of drug sensitivity.

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific ...

The overall goals of this combined In Vitro/In Silico Lung Cancer Model are to study the sensitivity of non-small cell lung cancer targeted therapies and to predict the efficacy of new substances. This 3-D model system allows the monitoring of cell specific responses to targeted treatments or direct combinations. The main advantage of this technique is that the covered with in Silico predictions can facilitate pre-clinical drug testing in a more physiological relevant environment.The implications of this technique extend towards the therapy of different tumor entities, as it can be used to optimize drug response predictions based on specific mutational backgrounds. Demonstrating the procedure will be, Franziska Schmitt, a research assistant, from our laboratory. To set up a dynamic culture, begin by culturing the tumor model under static culture conditions, in a cell crown at 37 degrees Celsius and 5%CO2, in a conventional incubator.After three days, assemble an Autoclaved Bioreactor, and insert a seeded, biological small intestine submucoso plus mucosa scaffold, into the system. Next, add 45 milliliters of RPMI, supplemented with the appropriate amount of FCS to a flask and connect the needle free sampling device to the tubing system between the Bioreactor and the flask of medium. Place the Bioreactor into the incubator and connect it to the pump to culture the tumor model, for an additional 14 days, with a constant medium flow of three milliliters per minute.To treat a static tumor model with, Gefitinib, on day 11 of culture in the conventional incubator, first use a pasteur pipet to remove the old medium, from the wells. Then, add one milliliter of freshly prepared, RPMI, supplemented with, FCS and Gefitinib, to the inside of each cell crown and 1.5 milliliters to the bottom of each well. To collect Elisa's samples from a dynamic culture, on days 11 through 14 of culture, attach the syringe to the sampling device and remove one milliliter of medium from the Bioreactor system.Then store the samples at, minus 80 degrees Celsius, until the Elisa is performed. To determine the number of Ki67 positive nuclei, first use an inverted microscope to obtain five DAPI and five Ki67 staining fluorescent images of 2D cultures at a 20 fold magnification of each condition, from non-overlapping sections of the specimen. To manually count the Ki67 positive nuclei, open emerged image in Fiji and select, Plugins, and Cell Counter, then, click initialize, select a counter type and click on each Ki67 positive nucleus.The number of clicked cells will appear next to the selected counter type. For automatic cell counting of the total number of nuclei in the 2D images, start the macro recorder, open a DAPI image, and click image, type and 8-bit, then click enhance contrast and set the saturated parameter to one and normalize. To sharpen the image, set the unsharp mask, using a radius of one and a mask of 60.To binderize the image, click auto threshold and choose the, Reny/Entropy method with white objects on black background. Then, click Plugins, followed by BioVoxxel, and watershed irregular feature, with an erosion radius of five to separate the cells. Click analyze, and analyze particles and set the size to 02 infinity and check summarize.The number of cells will be shown in the summary table as counts. Then, click create, to save the macro to allow automatic counting of nuclei, in further 2D cell culture images. After using know databases to generate a tumor network, open the appropriate network analysis software, click file and new, and input jove tumor models.Click ok, to generate a new network, then click, square closeup North and ok, to visualize the receptor in a double membrane. To visualize the nodes as rectangles, click generic protein, then EGFR, then Ok.To label the nodes in the network, right-click to change color and shape, and set the node color codes as indicated. To visualize the readout parameters as hexagons, select, phenotype, enter proliferation, and click ok.Then, right-click and select change color and shape again to set the color code to 255, 204, 204. To visualize the edges of node interactions, click state transition for activation arrows, and inhibition for blunted inhibition arrows. Then, click file and save as, and save the generated signaling network, as jove tumor models.xml.For steady state analysis of the system equilibrium, open the SQUAD software, click load network, and upload the jove tumor models. xml file. Click, run analysis.To write a simulation protocol, click advanced, and pertubator, then click edit protocol, to simulate the anit-EGFR resistance. Next, click, pertubation and select the standard initial state equals steady state four. Click add, to add the new constant pulse, then select the state parameter and set the target to FLIP, the time to zero, and the value to 4.Click, ok, and set the states for c-MET and Gefitinib, in the same way. To set a state value to the active EGFR node, click active node, then, click edit. Set the state to 8 and click ok.Click ok, to save the protocol. To display the specific curves, under the options panel, select EGF-EGFR, star EGF-EGFR, star Mek, caspases, PI3K, Gegitinib, apoptosis, proliferation, MEk inhibitor and PI3K inhibitor. Then, click initialize and run to start a complete simulation of the systems response.To simulate a combined PI3K and Mek inhibitor treatment, click edit protocol. Next, using the same node parameter as just demonstrated, click pertubation, followed by add, to add a new constant pulse. Then, select a state parameter, and set the target to PI3K inhibitor, the time to zero and the value to one, and click ok.Click add, again, to add another new constant pulse and under the state parameter, set the target to Mek inhibitor, set the time to zero and the value to one, and click ok, followed by ok again, to save the protocol. Under the options panel, using the same nodes for the graphical representation as just demonstrated, click initialize, and run to start a complete simulation of the systems response. Then, to finish the simulation, click reset.This 3D tumor test system, facilitates the determination of the proliferation index. And the quantification of apoptosis using M30 Elisa. In this representative experiment, for example, only EGFR mutated HCC827 tumor cells, incubated under static culture conditions, responded to epidermal growth factor inhibition.By the Tyrosine-kinase inhibitor, Gefitinib, as evident by the increase in apoptosis observed in the HCC827 tumor model system. Here representative images of static 3D tumor models, with or without, Epithelial-Mesenchymal transition inducer, TGF-beta-1 simulation, are shown. Note the increase in vimentin expression, in the treated tumor cell cultures.Under dynamic conditions, Gefitinib, exerts a robust effect on, EGFR mutated, HCC827 cells, as evidenced by the decrease of tissue-like structures, in the presence of the Tyrosine-kinase inhibitor, compared to the untreated controlled culture. In this graph, the simulation of the known drive in mutation, EGFR and c-MET co-activation, and increased proliferation rate, and a decreased apoptosis rate in HCC827 cells, over time, are observed, reflecting a Gefitinib induced resistance that correlates with activation of MEK and PI3K. Indeed, modeling of the combined PI3K and MEK inhibitors results in a reversal of the anti-EGFR resistance effect, a reduction in proliferation and the induction of apoptosis.Following this procedure, other methods like, Western Blot or can be performed to investigate downstream signaling pathways. Moreover, co-culture systems with different cell types may also be used to analyze the cross-talk between various cells in the tumor micro environment. This technique paves the way for researchers in the field of drug development to explore drug efficacy in Silico.After watching this video, you should have a good understanding of how to predict drug efficacy in stratified patient groups, by simulating signaling changes and analyzing the resulting system responses in different mutation and backgrounds.

a-combined-3d-tissue-engineered-vitroin-silico-lung-tumor-model-for

Research

JoVE Journal

Bioengineering

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 ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these procedures in basic research and preclinical drug development, it is important to develop protocols for automated generation and analysis under standardized conditions. Here, we present a technique to generate engineered heart tissue (EHT) from cardiomyocytes of different species (rat, mouse, human). The technique relies on the assembly of a fibrin-gel containing dissociated cardiomyocytes between elastic polydimethylsiloxane (PDMS) posts in a 24-well format. Three-dimensional, force-generating EHTs constitute within two weeks after casting. This procedure allows for the generation of several hundred EHTs per week and is technically limited only by the availability of cardiomyocytes (0.4-1.0 x 106/EHT). Evaluation of auxotonic muscle contractions is performed in a modified incubation chamber with a mechanical interlock for 24-well plates and a camera placed on top of this chamber. A software controls a camera moved on an XYZ axis system to each EHT. EHT contractions are detected by an automated figure recognition algorithm, and force is calculated based on shortening of the EHT and the elastic propensity and geometry of the PDMS posts. This procedure allows for automated analysis of high numbers of EHT under standardized and sterile conditions. The reliable detection of drug effects on cardiomyocyte contraction is crucial for cardiac drug development and safety pharmacology. We demonstrate, with the example of the hERG channel inhibitor E-4031, that the human EHT system replicates drug responses on contraction kinetics of the human heart, indicating it to be a promising tool for cardiac drug safety screening.

Here, we show the generation of human engineered heart tissue from induced pluripotent stem cells (hiPSC)-derived cardiomyocytes. We present a method to analyze contraction force and exemplary alteration of contraction pattern by the hERG channel inhibitor E-4031. This method shows high level of robustness and suitability for cardiac drug screening.

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional tissue. Tissue engineering holds great promise as a potential solution with its ability to integrate cells and signaling molecules into a 3-dimensional scaffold. Recent work with tissue engineered tracheal grafts (TETGs) has seen some success but their translation has been limited by graft stenosis, graft collapse, and delayed epithelialization. In order to investigate the mechanisms driving these issues, we have developed a mouse model for tissue engineered tracheal graft implantation. TETGs were constructed using electrospun polymers polyethylene terephthalate (PET) and polyurethane (PU) in a mixture of PET and PU (20:80 percent weight). Scaffolds were then seeded using bone marrow mononuclear cells isolated from 6-8 week-old C57BL/6 mice by gradient centrifugation. Ten million cells per graft were seeded onto the lumen of the scaffold and allowed to incubate overnight before implantation between the third and seventh tracheal rings. These grafts were able to recapitulate the findings of stenosis and delayed epithelialization as demonstrated by histological analysis and lack of Keratin 5 and Keratin 14 basal epithelial cells on immunofluorescence. This model will serve as a tool for investigating cellular and molecular mechanisms involved in host remodeling.

Graft stenosis poses a critical obstacle in tissue engineered airway replacement. To investigate cellular mechanisms underlying stenosis, we utilize a murine model of tissue engineered tracheal replacement with seeded bone marrow mononuclear cells (BM-MNC). Here, we detail our protocol, including scaffold manufacturing, BM-MNC isolation, graft seeding, and implantation.

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...

Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models is crucial both to better understand the pathological mechanisms of these diseases and identify new therapeutic targets and strategies. To be pertinent, an in vitro model must reproduce the pathological features of a human disease. However, in the context of neurodegenerative disease, a relevant in vitro model should provide neural cell replacement as a valuable therapeutic opportunity. 
Such a model would not only allow screening of therapeutic molecules but also can be used to optimize neural protocol differentiation [for example, in the context of transplantation in Parkinson's disease (PD)]. This study describes two in vitro protocols of 1) human glioblastoma development within a human neural organoids (NO) and 2) neuron dopaminergic (DA) differentiation generating a three-dimensional (3D) organoid. For this purpose, a well-standardized protocol was established that allows the production of size-calibrated neurospheres derived from human embryonic stem cell (hESC) differentiation. The first model can be used to reveal molecular and cellular events occurring during in glioblastoma development within the neural organoid, while the DA organoid not only represents a suitable source of DA neurons for cell therapy in Parkinson's disease but also can be used for drug testing.

This study introduces and describes protocols to derive two specific human neural organoids&#160;as a relevant and accurate model for studying 1) human glioblastoma development within human neural organoids exclusively in humans and 2) neuron dopaminergic differentiation generating a three-dimensional organoid.

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models ...

Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant technique with nutritional support. Recently, there have been many attempts at esophageal tissue engineering, but the success rate has been limited due to difficulty in early epithelization in the special environment of peristalsis. Here, we developed an artificial esophagus that can improve the regeneration of the esophageal mucosa and muscle layers through a two-layered tubular scaffold, a mesenchymal stem cell-based bioreactor system, and a bypass feeding technique with modified gastrostomy. The scaffold is made of polyurethane (PU) nanofibers in a cylindrical shape with a three-dimensional (3D) printed polycaprolactone strand wrapped around the outer wall. Prior to transplantation, human-derived mesenchymal stem cells were seeded into the lumen of the scaffold, and bioreactor cultivation was performed to enhance cellular reactivity. We improved the graft survival rate by applying surgical anastomosis and covering the implanted prosthesis with a thyroid gland flap, followed by temporary nonoral gastrostomy feeding. These grafts were able to recapitulate the findings of initial epithelialization and muscle regeneration around the implanted sites, as demonstrated by histological analysis. In addition, increased elastin fibers and neovascularization were observed in the periphery of the graft. Therefore, this model presents a potential new technique for circumferential esophageal reconstruction.

Esophageal reconstruction is a challenging procedure, and development of a tissue-engineered esophagus that enables regeneration of esophageal mucosa and muscle and that can be implanted as an artificial graft is necessary. Here, we present our protocol to generate an artificial esophagus, including scaffold manufacturing, bioreactor cultivation, and various surgical techniques.

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant ...