The authors would like to thank Jan Hansmann (Fraunhofer IGB, Stuttgart) for his technical support to develop bioreactors and the bioreactor incubator.

1. Decellularization of the BioVaSc
<ol>
	<li>Rinse the vascular system of the porcine jejunal segment via cannulated arterial access and the intestinal lumen with PBS-. Repeat until it is completely clean.</li>
	<li>Prepare a 200 mm diameter glass tank with 4 adapters and connect them via silicon tubes to the peristaltic pump (Ismatec). The pressure controlling unit can be monitored via a pressure sensor that is connected to a sterile disposable dome (see Figure 1).</li>
	<li>Fill reservoir bottles with decellularization (DZ) solution and check the tubing system for possible air bubbles.</li>
	<li>Connect the intestinal lumen with cable ties to the glass connectors for luminal flow. Pump 500 ml DZ solution into the red arterial access (Figure 1B) of the vascular system. Interrupt the pumping process every 15 min shortly to manually press out the entire intestinal lumen.</li>
	<li>During the decellularization process, the pressure of the buffer solution should be between 80 - 100 mm Hg, modeled after the natural blood pressure.</li>
	<li>Wash the BioVaSc with PBS- until it is free of cell remnants ("completely white").</li>
	<li>Fill the BioVaSc completely with DZ solution and incubate it submerge in DZ solution over night at 4 &deg;C on rocking shaker.</li>
	<li>Repeat step 1.6.</li>
	<li>Place the BioVaSc in DNase solution and incubate over night at 4 &deg;C on rocking shaker.</li>
	<li>Remove DNase solution and rinse with washing buffer.</li>
	<li>&gamma;-sterilization with 25 kGy</li>
</ol>
2. The Different Cell Types
2.1 Isolation of primary human dermal microvascular endothelial cells (mvECs) and fibroblasts
<ol>
	<li>Cut skin biopsy (preferably preputium) into strips of 2 - 3 mm width with scalpel and rinse them 3x with PBS- solution.</li>
	<li>Cover the tissue with Dispase solution and incubate it for 16 - 18 hr at 4 &deg;C.</li>
	<li>Separate epidermis from dermis with 2 tweezers and transfer both separately into petri dishes filled with PBS+.</li>
	<li>Rinse dermis strips 1x with Versene.</li>
	<li>Add 10 ml Trypsin/EDTA solution to the dermis strips and incubate it in the incubator for 40 min.</li>
	<li>Stop the enzyme reaction immediately with 1% FCS.</li>
	<li>Transfer skin strips to a petri dish filled with VascuLife and scratch out each strip with the scalpel 8x each side, adding a little pressure.</li>
	<li>Transfer the produced cell suspension via a cell strainer to a centrifuge tube and rinse cell strainer 3x with VascuLife.</li>
	<li>Centrifuge the tube at 1,200 x g for 5 min and resuspend the cell pellet with VascuLife.</li>
	<li>To isolate the fibroblasts, chop dermis strips into little pieces using the scalpel.</li>
	<li>Add 10 ml of collagenase solution to dermis pieces.</li>
	<li>Incubate it for 45 min in the incubator, then centrifuge it and carefully remove supernatant.</li>
	<li>Wash pellet 1x with DMEM + 10% FCS + % PenStrep, centrifuge it and carefully remove supernatant.</li>
	<li>Resuspend the pellet in culture medium and transfer it to a T75 culture flask to allow cells to grow out of the tissue.</li>
</ol>
2.2 Tumor Cell Line S462
The tumor cell line S462 (kindly provided by Dr. Nikola Holtkamp, Charit&eacute; University Medicine Berlin) was generated from a malignant peripheral nerve sheath tumor of a female patient with the hereditary tumor predisposition syndrome neurofibromatosis type 1 9. S462 are cultured in DMEM supplemented with 10% FCS. Medium has to be changed every 2 - 3 days. Once a week the cells have to be split.
3. Tumor Test System: Static Culture Conditions Compared with Dynamic Culture In Bioreactor Systems
<ol>
	<li>Cut the tubular SIS-Muc open on one side and fix it between two metal rings (cell crowns, 10 mm diameter, self constructed). Cover the SIS-Muc in cell culture medium overnight.</li>
	<li>Seed isolated cells in defined cell numbers (see below) on one or both sides of the SIS (mono- or co-culture set-up).
	<ol>
		<li>Seed 8,000 cells/cm2 of primary mvECs in a total volume of 100 &mu;l onto the basolateral surface of the SIS (former serosa). 3 hr later fill the well with medium to ensure a submersed culture.</li>
		<li>Allow endothelial cells to adhere for 3 days. Flip the static culture system by 180&deg; and transfer it to a 12-well plate.</li>
		<li>Seed a mixture of primary dermal fibroblasts (8,000 cells/cm2) and tumor cells (15,000 cells/cm2) within a total volume of 500 &mu;l on the apical surface of the SIS (the side of the former lumen).</li>
		<li>Allow cells to adhere for 3 hr and fill the well with medium (submersed culture, medium: 50% Vasculife + 50% DMEM supplemented with 10% FCS).</li>
		<li>The tumor test system is cultured under static conditions at 37 &deg;C, 5% CO2 in the incubator for additional 14 days. Change culture medium every 2 - 3 days.</li>
	</ol>
	</li>
	<li>For the dynamic culture, fix the SIS-Muc between two metal rings and seed primary mvECs as described in 3.2.1. After 3 days remove the SIS-Muc from the metal rings and insert the membrane into the flow reactor (see Figures 2C and 2D). Apply the primary dermal fibroblasts and tumor cells with a syringe and cannula onto the matrix in the bioreactor, and allow the cells to adhere for 3 hr before filling the bioreactor system with culture medium.</li>
	<li>On the following day dynamic culture conditions with constant medium flow (3.8 ml/min, 37 &deg;C, and 5% CO2) can be initiated. The dynamic culture is maintained for 14 days, culture medium is changed after 7 days.</li>
	<li>The dynamic test setup is cultured in a self-constructed incubator that provides media flow through a pump and the necessary temperature and CO2 content.</li>
</ol>
4. Characterization Methods for Analysis
4.1 Fixing and paraffin-embedding of the seeded collagenous matrix
<ol>
	<li>For (immuno-) histological characterizations remove the culture medium and fix the tissue with 4% paraformaldehyde for 2 hr.</li>
	<li>Remove 4% paraformaldehyde and transfer the SIS from the metal insert to a tissue embedding cassette. Water the tissue to remove remaining fixative and dehydrate for paraffin infiltration.</li>
	<li>Before embedding in a paraffin block, cut the SIS in 2 - 3 slices and place them in a paraffin-filled metal base mold so that the cut surfaces face downwards. Add the tissue cassette on top of the mold as a backing.</li>
	<li>Cut 5 &mu;m slices and float them on a 40 &deg;C water bath for straightening, then mount slides onto suitable glass slides. Uncoated slides are used for H&amp;E stains, polylysine-coated slides for immunohistological staining to improve attachment. Let slices dry thoroughly.</li>
	<li>Melt paraffin, remove it with xylene and rehydrate slices for following staining.</li>
</ol>
4.2 Staining
<ol>
	<li>The rehydrated slices can be stained with Hematoxylin/Eosin as a standardized overview staining.</li>
	<li>For immunhistological staining, the fixed and paraffin-infiltrated tissue slices must undergo an antigen retrieval to allow antibodies to recognize and bind to their specific epitopes. Therefore, deparaffinized and rehydrated slides are placed in a steam cooker with heated citrate buffer (pH 6.0) for 20 min.</li>
	<li>Transfer slides to washing buffer (0.5 M TBS buffer + 0.5% Tween) and circle slices with a PAP pen to minimize the required volume for staining solutions.</li>
	<li>Place the slide in a moisture chamber. To secure a specific horseradish peroxidase-mediated visualization of antigen-antibody-binding, the endogenous peroxidase must be saturated with 3% hydrogen peroxide.</li>
	<li>Primary antibody dilutions are applied to the slices, incubated for 1 hr at RT and carefully washed off with washing buffer.</li>
	<li>For detection of specific antigen-antibody bindings the Ultra Vision Quanto Detection System HRP DAB (Thermo Scientific) is used according to the recommended protocol.</li>
	<li>Nuclei are counterstained with Hematoxylin for 1 min.</li>
	<li>Slides are mounted with an aqueous medium, dried, and imaged using an inverse microscope.</li>
</ol>

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	<li>Schenke-Layland, K., Nerem, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+human+tissue+models--moving+towards+personalized+regenerative+medicine.">In vitro human tissue models--moving towards personalized regenerative medicine.</a> Adv. Drug Deliv. Rev. 63, 195-196 (2011).</li><li>Pusch, J., Votteler, 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=The+physiological+performance+of+a+three-dimensional+model+that+mimics+the+microenvironment+of+the+small+intestine.">The physiological performance of a three-dimensional model that mimics the microenvironment of the small intestine.</a> Biomaterials. 32, 7469-7478 (2011).</li><li>Schanz, J., Pusch, J., Hansmann, J., Walles, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularised+human+tissue+models:+a+new+approach+for+the+refinement+of+biomedical+research.">Vascularised human tissue models: a new approach for the refinement of biomedical research.</a> J. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+of+a+vascularized+scaffold+for+artificial+tissue+and+organ+generation.">Engineering of a vascularized scaffold for artificial tissue and organ generation.</a> Biomaterials. 26, 6610-6617 (2005).</li><li>Linke, K., Schanz, J., Hansmann, J., Walles, T., Brunner, H., Mertsching, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+liver-like+tissue+on+a+capillarized+matrix+for+applied+research.">Engineered liver-like tissue on a capillarized matrix for applied research.</a> Tissue Eng. 13, 2699-2707 (2007).</li><li>Holtkamp, N., Atallah, I., 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=MMP-13+and+p53+in+the+progression+of+malignant+peripheral+nerve+sheath+tumors.">MMP-13 and p53 in the progression of malignant peripheral nerve sheath tumors.</a> Neoplasia. 9, 671-677 (2007).</li><li>Weaver, V. 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Authors have nothing to disclose.

When comparing 2D and 3D culture systems in tumor research, 3D systems, despite being the more expensive approach, have proven to mimic the conditions in biological microenvironments better. It could be shown that some tumor cells grow much slower in a 3D culture than in a common 2D culture 12, which is in accordance to the situation in a real tumor. Bissell and coworkers showed in their work that the behavior of carcinogenic breast cells reflects the in vivo situation, including cell morphology and signaling, more accurately when a 3D culture within a matrix offers cell-ECM interactions. Furthermore, they emphasized the importance of the extracellular environment in 3D by demonstrating that changes in the environmental interactions led to the reversion of the malignant cells to a normal phenotype. Additionally and most importantly, these outcomes could also be confirmed in in vivo animal models 10,11.
The direct comparison of in vivo animal experiments and in vitro tissue models reveals advantages and drawbacks in both systems. One advantage of in vitro models is the permission of a much better real-time or fixed imaging by microscopy. A limitation is that they mimic static or short-term conditions, whereas in vivo systems often progress. The current lack of vasculature and normal transport of small molecules, host immune responses, and other cell-cell interactions are further disadvantages of in vitro models 12. Therefore, 3D in vitro systems as presented in this study offer a promising addition to animal experiments. They provide a better comparability to the human organism and therefore minimize experimental misinterpretations. Biomimetic in vivo model systems will hence become more relevant to study how cancer and metastatic spread is dependent on microenvironmental conditions that regulate tumorigenesis 11.
Our study shows that the 3D environment provided by the SIS-Muc leads to a more tumor-like tissue formation of cells, which is not observed in the common 2D cell culture (see Figure 3A). Moreover, the use of primary cells derived from tumor biopsies is a very important step towards personalized medicine, a discipline that aims at identifying the best treatment depending on a patient's individual needs. Incorporating primary patient-specific tumor cells isolated from biopsy material will allow in vitro testing of therapeutic strategies. Such test systems will make it possible to investigate different drugs and combinations thereof in a time- and cost-saving high-throughput screening. Additionally, the integration of tumor-associated stromal cells as shown in this study is important for the personalized approach, since a tumor's microenvironment influences tumor progression 13 and might prove as suitable therapeutic target.
Alternatively to a personalized approach, our tumor model can be modified to serve as a generalized tumor test system by the incorporation of established tumorigenic cell lines. This is a promising adaptation for basic research purposes. For both drug testing approaches the presence of a vascular structure is required to test the distribution and uptake of therapeutic substances. The SIS-Muc matrix allows the basolateral seeding with primary mvEC for barrier uptake studies, the reseeding of the preserved vascular structures of the BioVaSc will further improve the study of drug delivery.
In order to create tissue models, a 3D biodegradable matrix can be used as framework for a co-culture of different cell types 14. The use of such 3D matrices is often limited by the absence of a functional vascularization. This problem can be solved by the use of the BioVaSc, which offers preserved blood vessel structures, which can be reseeded with endothelial cells. Furthermore, the BioVaSc provides extracellular components, which ensure the adhesion of the cells and facilitate tissue differentiation. It also enables the long-time tissue specific function of bioartificial 3D tissues 7,8,15. The prerequisite for the engineering of functional vascular substitutes is the mimicking of human physiological and biomechanical conditions. Therefore, bioreactor systems, which can implement these requirements in vitro, are of extreme interest for creating biological tumor models.
The combination of the BioVaSc, the bioreactor technology and co-culturing of different cell-types is a very promising method to generate vascularized tumor tissues, which will allow the study of mechanisms relevant for cancer progression such as angiogenesis and metastasis. We see such tumor models as a promising approach for complementing animal studies by providing an equivalent to the human tumor physiology.

New pharmaceuticals must be validated in regard to their quality, safety, and efficacy before market authorization. To date, animal experiments are the standard method for drug testing and validation. However, due to species-specific differences, animal experiments often do not comprehensively evaluate the effect of the compounds in humans 1. For this reason, it is important to generate human tissue models that can be used for in vitro tests of new drugs and substances.
One of the focuses of our group is the creation of in vitro test models with our biological vascularized scaffold (BioVaSc) 2,3. The BioVaSc can be used as a static or dynamic 3D matrix system. For static culture, the decellularized porcine jejunal segment (Small Intestinal Submucosa SIS-Muc) is placed in a metal insert for cell reseeding. Various cells, such as cancer and endothelial cells can be cultured on the scaffold.
For dynamic culture, the BioVaSc is attached to a bioreactor system that applies flow throughout the vasculature or across the surface of the scaffold. Current bioreactors implement biological, mechanical, or electrical stimuli that act upon the differentiation or proliferation of cells 4. For bioreactors in the field of Tissue Engineering, the basic concept is to simulate the conditions in the human body. Wherein, cells are provided a natural environment in which they can interact with each other and their surrounding extracellular matrix. For the production of in vitro test systems or transplants, the ability to mimic the natural environment of cells with an appropriate carrier structure and bioreactor system is critical 5. Therefore, more complex and technically demanding devices must be developed in order to fulfill these tasks 6.
It is furthermore possible to use our scaffold for the establishment of a vascularized model due to the preserved tubular structures, which include the feeding artery, vein, and the connecting capillary bed. All porcine cells need to be removed by chemical, mechanical and enzymatic decellularization, and the scaffold gamma-sterilized. The restored tubular vascular structures can subsequently be reseeded with human microvascular endothelial cells using a recirculation perfusion bioreactor 7, which mimics the biomechanical and/or biochemical parameters such as pH, temperature, pressure, nutrient supply and waste removal 6. The re-endothelialization of the tubular structures creates a human blood vessel equivalent within the collagenous scaffold 3,7. In the following step, the surface of the former lumen (mucosa) can be seeded with primary human cells to establish co-cultures 3,7,8.
In this study a 3D tumor test system is set up by co-culturing a tumor cell line with primary stromal cells under static and dynamic conditions on the SIS-Muc.

<table border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Collagenase solution</td>
			<td>SERVA</td>
			<td>17454</td>
			<td>(500 U/ml)</td>
		</tr>
		<tr>
			<td>Dispase solution</td>
			<td>Gibco</td>
			<td>17105-041</td>
			<td>(2.0 U/ml)</td>
		</tr>
		<tr>
			<td>DMEM, high-glucose</td>
			<td>PAA</td>
			<td>G0001,3010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>ROCHE</td>
			<td>10104159001</td>
			<td>200 mg solved in 500 ml PBS+ + 1% PenStrep</td>
		</tr>
		<tr>
			<td>DZ solution</td>
			<td>Roth</td>
			<td>3484.2</td>
			<td>34 g Sodium Desoxychelate, in 1 L Ultra-pure water</td>
		</tr>
		<tr>
			<td>FCS</td>
			<td>LONZA</td>
			<td>DE14-801F</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>IHC-Kit DCS SuperVision 2 HRP</td>
			<td>DCS</td>
			<td>PD000KIT</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>medical pressure transducer</td>
			<td>MEMSCAP</td>
			<td>SP844</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>monoclonal mouse anti-human Von Willebrand Factor</td>
			<td>DAKO Cytomation</td>
			<td>M0616</td>
			<td>Clone F8/86 0.12 &mu;g/ml</td>
		</tr>
		<tr>
			<td>mouse monoclonal anti-human p53</td>
			<td>DAKO Cytomation</td>
			<td>IS616</td>
			<td>Clone DO-7 ready-to-use</td>
		</tr>
		<tr>
			<td>peristaltic pump</td>
			<td>Ismatec</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>sterile disposable dome</td>
			<td>MEMSCAP</td>
			<td>844-28</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trypsin / EDTA solution</td>
			<td>PAA</td>
			<td>L11-003</td>
			<td>0,05%</td>
		</tr>
		<tr>
			<td>VascuLife (VEGF-Mv)</td>
			<td>Lifeline</td>
			<td>LL-0003</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Versene</td>
			<td>Gibco</td>
			<td>15040-033</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

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.

As shown in Figure 1B, we decellularized the porcine jejunal segment (about 2 m in length and 20 mm in diameter) with preserved tubular structures of the capillary network. After chemical, enzymatic and mechanical decellularization, we obtained a collagen I/III scaffold, which can be used for 3D cell culture. A Feulgen test was performed to demonstrate the purity (no DNA remnants) of the matrix (data not shown).
Figures 2A and 2B show the static culture of SIS-Muc secured by the cell crowns. We fixed the SIS-Muc in an in-house designed bioreactor (Figure 2C) for dynamic culture. Figure 2D illustrates the simulated dynamic flow through the chamber of the bioreactor. The bioreactor is placed in a self-constructed incubator system and connected with a peristaltic pump. This setup allows a dynamic culture with either pressure-regulated pulsatile flow or constant flow.
Figure 3 gives an overview of the statically cultured S462 tumor cell line in 2D monoculture (Figure 3A) and in 3D coculture (Figures 3B-3D). Figure 3B shows the triple culture of tumor cells S462 and primary fibroblasts on the apical side of SIS-Muc (former inner lumen side) and mvEC on the basolateral side (former serosa side). The identification of different cell types is possible by staining cell-type specific markers, such as von Willebrand factor to label mvEC (Figure 3C). The p53-positive S462 cells can be distinguished from the p53-negative primary fibroblasts (Figure 3D) and the 3D distribution of cells can be analyzed. Figure 4 shows stainings equivalent to Figure 3 of the dynamically cultured triple culture.
<img alt="Figure 1" src="/files/ftp_upload/50460/50460fig1.jpg" /> 
 Figure 1. Decellularization set-up. (A) Bioreactor and pump set-up for decellularizing the BioVaSc, monitored by a PC. (B) Decellularized BioVaSc in glass tank. The lumen and the arterial inlet are connected to the adapters. .
<img alt="Figure 2" src="/files/ftp_upload/50460/50460fig2.jpg" /> 
 Figure 2. Over view of the different culture set-ups. (A) CAD section view of static culture system in Microtiter well plate, (B) metal inserts for static culture, (C) medium flow simulation (velocity field [m/sec]) for dynamic culture of the upper lid of the flow bioreactor, (D) flow bioreactor for dynamic culture connected to the peristaltic pump. <a href="https://www.jove.com/files/ftp_upload/50460/50460fig2large.jpg" target="_blank"> Click here to view larger figure</a>
<img alt="Figure 3" fo:src="/files/ftp_upload/50460fig3highres.jpg" fo:content-width="5.5in" src="/files/ftp_upload/50460/50460fig3.jpg" /> 
 Figure 3. Overview of the immunohistological characterization of the static 3D tumor model. (A) Hematoxylin stain of the statically cultured 2D mono culture of S462 cells on a Permanox slide, (B) H&amp;E stain of the statically cultured 3D triple culture, arrows mark endothelial cells, (C) immunohistological staining for von Willebrand factor, (D) immunohistological staining for p53. <a href="https://www.jove.com/files/ftp_upload/50460/50460fig3large.jpg" target="_blank"> Click here to view larger figure</a>
<img alt="Figure 4" fo:src="/files/ftp_upload/50460fig4highres.jpg" fo:content-width="6in" src="/files/ftp_upload/50460/50460fig4.jpg" /> 
 Figure 4. Overview of the immunohistological characterization of the dynamic 3D tumor model. (A) H&amp;E stain of the dynamically cultured 3D triple culture, arrows mark endothelial cells, (B) immunohistological staining for von Willebrand factor, (C) immunohistological staining for p53. <a href="https://www.jove.com/files/ftp_upload/50460/50460fig4large.jpg" target="_blank"> Click here to view larger figure</a>

Watch this Scientific Journal Video about Tissue Engineering of a Human 3D in vitro Tumor Test System at JoVE.com

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.

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 ...

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Research

JoVE Journal

Bioengineering

21.2K Views.  University Hospital Würzburg. 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.

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

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.

Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

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. ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

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.

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.

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 ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

Force System with Vertical V-Bends: A 3D In Vitro Assessment of Elastic and Rigid Rectangular Archwires

A proper understanding of the force system created by various orthodontic appliances can make treatment of patients efficient and predictable. Reducing the complicated multi-bracket appliances to a simple two-bracket system for the purpose of force system evaluation will be the first step in this direction. However, much of the orthodontic biomechanics in this regard is confined to 2D experimental studies, computer modeling/analysis or theoretical extrapolation of existing models. The objective of this protocol is to design, construct and validate an in vitro 3D model capable of measuring the forces and moments generated by an archwire with a V-bend placed between two brackets. Additional objectives are to compare the force system generated by different types of archwires among themselves and to previous models. For this purpose, a 2 x 4 appliance representing a molar and an incisor has been simulated. An orthodontic wire tester (OWT) is constructed consisting of two multi-axis force transducers or load cells (nanosensors) to which the orthodontic brackets are attached. The load cells are capable of measuring the force system in all the three planes of space. Two types of archwires, stainless-steel and beta-titanium of three different sizes (0.016 x 0.022 inch, 0.017 x 0.025 inch and 0.019 x 0.025 inch), are tested. Each wire receives a single vertical V-bend systematically placed at a specific position with a predefined angle. Similar V-bends are replicated on different archwires at 11 different locations between the molar and incisor attachments. This is the first time an attempt has been made in vitro to simulate an orthodontic appliance utilizing V-bends on different archwires.

Force System with Vertical V-Bends: A 3D In Vitro Assessment of Elastic and Rigid Rectangular Archwires

The method presented here is designed to construct and validate an in vitro 3D model capable of measuring the force system generated by different archwires with V-bends placed between two brackets. Additional objectives are to compare this force system with different types of archwires and to previous models.

A proper understanding of the force system created by various orthodontic appliances can make treatment of patients efficient and predictable. Reducing ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart muscle, the myocardium, is notoriously difficult to accomplish in vitro. Mainly, obstacles lie in the need for human cardiomyocytes (CMs) that are either adult or exhibit adult-like phenotypes and can successfully replicate the myocardium&#39;s cellular complexity and intricate 3D architecture. Unfortunately, due to ethical concerns and lack of available primary patient-derived human cardiac tissue, combined with the minimal proliferation of CMs, the sourcing of viable human CMs has been a limiting step for cardiac tissue engineering. To this end, most research has transitioned toward cardiac differentiation of human induced pluripotent stem cells (hiPSCs) as the primary source of human CMs, resulting in the wide incorporation of hiPSC-CMs within in vitro assays for cardiac tissue modeling.
Here in this work, we demonstrate a protocol for developing a 3D mature stem cell-derived human cardiac tissue within a microfluidic device. We specifically explain and visually demonstrate the production of a 3D in vitro anisotropic cardiac tissue-on-a-chip model from hiPSC-derived CMs. We primarily describe a purification protocol to select for CMs, the co-culture of cells with a defined ratio via mixing CMs with human CFs (hCFs), and suspension of this co-culture within the collagen-based hydrogel. We further demonstrate the injection of the cell-laden hydrogel within our well-defined microfluidic device, embedded with staggered elliptical microposts that serve as surface topography to induce a high degree of alignment of the surrounding cells and the hydrogel matrix, mimicking the architecture of the native myocardium. We envision that the proposed 3D anisotropic cardiac tissue-on-chip model is suitable for fundamental biology studies, disease modeling, and, through its use as a screening tool, pharmaceutical testing.

The goal of this protocol is to explain and demonstrate the development of a three-dimensional (3D) microfluidic model of highly aligned human cardiac tissue, composed of stem cell-derived cardiomyocytes co-cultured with cardiac fibroblasts (CFs) within a biomimetic, collagen-based hydrogel, for applications in cardiac tissue engineering, drug screening, and disease modeling.

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart ...