The authors acknowledge support from NSF grant CBET-0909678 and funding from the GW Center for Biomimetics and Bioinspired Engineering (COBRE). We thank the students, Mr. Christopher Popma, Ms. Leanne Penna, Ms. Shannon Callahan, Mr. Shadman Hussain, Mr. Mohammed R. Najjari, and Ms. Jessica Hinke for help in the laboratory and Mr. Mathieu Barraja for assisting in CAD drawings.

<ol>
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No conflicts of interest declared.

The protocol presented in this paper describes the acquisition of high fidelity experimental data using particle image velocimetry technique (PIV) and coherent structure detection methods, viz., continuous wavelet transforms, <img alt="Equation 1" src="/files/ftp_upload/51288/51288eq1.jpg" />, suited for identification of vortex and shear-dominated flows. Analysis of experimental data from physiological inflows in the presence of an idealized &#34;Type IV&#34; fracture reveals that secondary flow structures with complica...

Secondary flow structures are vortical flow patterns that occur in internal flow geometries with curvatures such as curved pipes and channels. These vortical structures arise due to the combined action of centrifugal forces, adverse pressure gradients and inflow characteristics. In general, secondary flow structures appear in planar cross-sections of curved pipes as symmetric Dean-type vortices under steady inflow and, symmetric Dean- and Lyne-type vortices under oscillatory inflow conditions 1 - 3. Secondary flow morphologies are greatly affected by pulsatility and multiple harmonics of pulsatile, physiological inflow conditions. These structures acquire markedly different size-strength-shape characteristics compared to non-physiological (steady and oscillatory) flows 1 - 6. Atherosclerotic lesion development in arteries is affected by the existence of high frequency shear oscillations in regions experiencing low mean shear 27, 28. Secondary flow structures may influence the progress of diseases such as atherosclerosis and possibly, mediate the endothelial response due to pulsatile blood flow by altering wall shear stresses and exposure times of blood-borne particles.
A common treatment to atherosclerosis, a complication resulting in narrowing of arteries by obstructive lesions, is the implantation of stents. Stent fractures are structural failures of implanted stents that lead to further medical complications such as in-stent restenosis (ISR), stent thrombosis and aneurysm formation 9 - 13. Stent fractures have been categorized into various failure &#34;Types I-to-IV&#34;, wherein &#34;Type IV&#34; characterizes the highest clinical severity and is defined as the complete transverse fracture of stent struts along with linear displacements of the stent fragments 14. The protocol presented in this study describes an experimental method of visualization of secondary flow structures downstream of an idealized &#34;Type IV&#34; stent fracture in a curved artery model.
The suggested protocol has the following four essential features:
Design and fabrication of laboratory-scale stent models: Geometric description of stents can be associated with a set of self-expandable spirals (springs or helices) intertwined using Nitinol (an alloy of nickel and titanium) wires 29. The length of the stent and its strut diameter depend on the length scale of arterial lesions encountered during clinical implantation 5. Parametric variation of strut diameter and the rising of the winding (or pitch) leads to stents of various geometric configurations. A summary of stent design parameters chosen for 3D printing are presented in Table 1.
Preparation of a blood analog working fluid matched with kinematic viscosity of blood and refractive index of the test section: Optical access to the curved artery test section is required in order to make non-invasive velocity measurements. Accordingly, a Newtonian blood-mimicking working fluid with the refractive index of the vascular model and ideally, a dynamic viscosity, matching human blood is used to obtain accurate blood flow measurements 16 - 18, 30. The working fluid used in this study was reported by Deutsch et al. (2006), that comprised of 79% saturated aqueous sodium iodide (NaI), 20% pure glycerol, and 1% water (by volume)16.
Experimental arrangement for the detection of coherent secondary flow structures using a two-component, two-dimensional particle image velocimetry (2C-2D PIV): Experiments were designed to acquire phase-averaged secondary flow velocity data at various planar cross-sectional locations downstream of a combination of straight and curved stent sections embodying an idealized &#34;Type IV&#34; stent fracture 5, 6, 9, 14. The protocol-steps pertaining to the acquisition of secondary flow velocity fields using particle image velocimetry (PIV) technique involves a PIV system that comprises of a laser (light sheet) source, optics to focus and illuminate the regions of flow, a special cross-correlation charge coupled device (CCD-sensor or camera) and tracer particles to be illuminated by the light sheet within a short time interval (&#916;t; see Table 4) 31, 32.
The steps in the protocol assume the following: First, a calibrated, experimental set-up of a two-component, two-dimensional (2C-2D) PIV system that evaluates images by double-frame, single-exposure recordings. Second, the 2C-2D PIV system calculates the mean displacements of tracer particles by performing cross-correlation between two image frames acquired during each recording. A brief summary of PIV specifications and image acquisition software is presented in the materials and equipment table. Third, all safety precautions needed to operate the laser are followed by trained laboratory personnel according to the guidelines provided by the host institution. The authors suggest Refs. 31 and 32 for a holistic understanding of the implementation, functionality and application of PIV technique in aero-, hydro- and microfluid dynamics, correlation peak detection and displacement estimation, material and density of tracer particles and, measurement noise and accuracy. Also note that the laser and camera can be controlled by the PIV data acquisition computer (Figure 3A) and data processing software.
Data acquisition and post-processing for coherent structure detection: Phase-averaged secondary flow velocity measurements using a 2C-2D PIV were generated using the protocol description that follows. Post-processing of the data involved coherent secondary flow structure detection using the following three methods: continuous wavelet transforms, <img alt="Equation 1" src="/files/ftp_upload/51288/51288eq1.jpg" /> 5, 6, 19 - 24, 26.
The authors note that the velocity gradient tensor is essentially, a 3 x 3 matrix, 
<img alt="Equation 2" src="/files/ftp_upload/51288/51288eq2.jpg" />.
The protocol presents a method of acquiring two-dimensional experimental measurements (from 2C-2D PIV technique). Therefore, full experimental access to the velocity gradient tensor will not be attainable using this method. The velocity gradient tensor for each pixel <img alt="Equation 3" src="/files/ftp_upload/51288/51288eq3.jpg" /> of the PIV image <img alt="Equation 4" src="/files/ftp_upload/51288/51288eq4.jpg" /> should be a 2 x 2 matrix, <img alt="Equation 5" src="/files/ftp_upload/51288/51288eq5.jpg" />. The z-component vorticity <img alt="Equation 6" src="/files/ftp_upload/51288/51288eq6.jpg" /> for each pixel <img alt="Equation 7" src="/files/ftp_upload/51288/51288eq7.jpg" /> is computed using the anti-symmetric part of the velocity gradient tensor <img alt="Equation 8" src="/files/ftp_upload/51288/51288eq8.jpg" />. The result will be a 2D array of vorticity <img alt="Equation 9" src="/files/ftp_upload/51288/51288eq9.jpg" /> that can be visualized in a contour plot. The authors strongly suggest Ref. 25 for an eloquent discussion experimental access to the velocity gradient tensor toward enhancing the knowledge of vorticity dissipation, strain rates and coherent structure detection. Furthermore, the authors do not attempt to explore the inter-relationships between the aforementioned coherent structure detection methods and suggest Ref. 23, 24 for a comprehensive discussion on that subject.
The focus of the steps in the protocol is the quantitative identification of secondary flow (vortical) structures (also known as coherent structures). Three methods of coherent structure detection viz., <img alt="Equation 10" src="/files/ftp_upload/51288/51288eq10.jpg" /> and wavelet transformed vorticity <img alt="Equation 11" src="/files/ftp_upload/51288/51288eq11.jpg" /> are applied to velocity field data toward detection of multi-scale, multi-strength occurrences of secondary flow structures downstream of the idealized &#34;Type IV&#34; stent fracture.
The <img alt="Equation 12" src="/files/ftp_upload/51288/51288eq12.jpg" />, defines a vortex as a spatial region where the Euclidean norm of the vorticity tensor dominates that of the rate of strain 19, 23, 24.The velocity gradient matrix is decomposed into symmetric (strain rate) and anti-symmetric (rotation) parts. Eigenvalues of strain rate matrix are computed; <img alt="Equation 13" src="/files/ftp_upload/51288/51288eq13.jpg" />. Norm of the strain rate is then calculated; <img alt="Equation 14" src="/files/ftp_upload/51288/51288eq14.jpg" />. Vorticity is computed from the anti- symmetric part. Enstrophy or square of z-component vorticity, <img alt="Equation 15" src="/files/ftp_upload/51288/51288eq15.jpg" />) is then computed. The <img alt="Equation 16" src="/files/ftp_upload/51288/51288eq16.jpg" /> is finally computed; <img alt="Equation 17" src="/files/ftp_upload/51288/51288eq17.jpg" />. A contour plot of the entire set of <img alt="Equation 18" src="/files/ftp_upload/51288/51288eq18.jpg" /> with iso-regions of <img alt="Equation 19" src="/files/ftp_upload/51288/51288eq19.jpg" />, will indicate secondary flow structures 19. 
The <img alt="Equation 20" src="/files/ftp_upload/51288/51288eq20.jpg" />, also known as &#39;swirling strength&#39; is a vortex identification method performed by critical-point analysis of the local velocity gradient tensor and its corresponding eigenvalues 20 - 24. Eigenvalues of the velocity gradient tensor at each pixel&#160;<img alt="Equation 21" src="/files/ftp_upload/51288/51288eq21.jpg" /> are computed. The eigenvalues should be of the form, <img alt="Equation 22" src="/files/ftp_upload/51288/51288eq22.jpg" />. A contour plot of <img alt="Equation 23" src="/files/ftp_upload/51288/51288eq23.jpg" /> with iso-regions of <img alt="Equation 24" src="/files/ftp_upload/51288/51288eq24.jpg" /> will indicate secondary flow structures 20 - 22.
Wavelet transform method utilizes an analyzing function (or wavelet) that has smoothness in physical and spectral spaces, is admissible (or has zero mean) and has a finite <img alt="Equation 25" src="/files/ftp_upload/51288/51288eq25.jpg" />5, 6, 26. By convolving a dilated or contracted wavelet with a 2D vorticity field, wavelet transformed vorticity&#160;<img alt="Equation 26" src="/files/ftp_upload/51288/51288eq26.jpg" /> field is generated comprising of coherent structures with a wide range of scales and strengths 5, 6, 26. Shannon entropy of the 2D wavelet-transformed vorticity field is computed to estimate the optimal wavelet scale at which all the coherent structures are adequately resolved. This entropy estimation involves a set of probabilities <img alt="Equation 27" src="/files/ftp_upload/51288/51288eq27.jpg" /> for each pixel <img alt="Equation 21" src="/files/ftp_upload/51288/51288eq21.jpg" /> such that&#160; <img alt="Equation 28" src="/files/ftp_upload/51288/51288eq28.jpg" />, the normalized square modulus of the vorticity associated with the pixel at location m, n 5, 6. The procedural steps are presented graphically in Figure 6. The restrictions placed on the choice of the wavelet are presented in detail in Ref. 26. This protocol step describes the procedure for coherent structure detection using a 2D Ricker wavelet. The justification for the use of this wavelet for vortical pattern matching is presented in Ref. 5, 6 and the pertinent references cited therein.

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			<td height="21" style="height:21px;width:216px;">VELP scientifica - ESP stirrer&#160;</td>
			<td style="width:155px;">VELP Scientifica</td>
			<td>F206A0179</td>
			<td style="width:208px;">Magnetic stirrer</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ohaus Scout Pro SP 601&#160;</td>
			<td style="width:155px;">The Lab Depot</td>
			<td>SP4001</td>
			<td style="width:208px;">Weigh scale</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Refractometer</td>
			<td>Atago</td>
			<td style="width:119px;">PAL-RI</td>
			<td style="width:208px;">Toward measurement of refractive index of blood-analog fluid</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Beakers, pipettes, syringes and spatula</td>
			<td style="width:155px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">CLS710110,&#160; CLS10031L, CLS71015, CLS71011 Z193216</td>
			<td style="width:208px;">Toward handling materials required for blood-analog solution preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Iodide</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td>383112-2.5KG&#160;</td>
			<td style="width:208px;">Crystalline</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycerol</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td>G5516-1L</td>
			<td style="width:208px;">Liquid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Deionized Water</td>
			<td style="width:155px;">-</td>
			<td style="width:119px;">-</td>
			<td style="width:208px;">Liquid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium thiosulfate anhydrous</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td>72049-250G</td>
			<td style="width:208px;">Powder</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PIV Recording medium</td>
			<td style="width:155px;">&#160;LaVision&#160;</td>
			<td style="width:119px;">Imager Intense 10Hz</td>
			<td style="width:208px;">PIV Image acquisition CCD camera</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">PIV Illumination source</td>
			<td style="width:155px;">New Wave Research</td>
			<td style="width:119px;">Solo III-15</td>
			<td style="width:208px;">PIV Laser source, Nd:YAG laser, 532 nm, dual pulse 70 mJ/pulse</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PIV Imaging software</td>
			<td style="width:155px;">&#160;LaVision&#160;</td>
			<td style="width:119px;">DaVis 7.2&#160;</td>
			<td style="width:208px;">PIV data acquisition and instrument control&#160;</td>
		</tr>
		<tr height="211">
			<td height="211" style="height:211px;width:216px;">PIV Seeding material</td>
			<td style="width:155px;">Thermo-scientific&#160;&#160;</td>
			<td style="width:119px;">Flouro-Max</td>
			<td style="width:208px;">Red fluorescent polymer microspheres (&#8776; 7 &#181;m); Dry dyed polystyrene (DVB) fluorescent microspheres emit bright and distinct colors when illuminated by the light of shorter&#160; wavelengths than the emission wavelength.&#160;</td>
		</tr>
	</tbody>
</table>

experimental investigation of secondary flow structures downstream of a model type iv stent failure in a 180&#176; curved artery test section

The arterial network in the human vasculature comprises of ubiquitously present blood vessels with complex geometries (branches, curvatures and tortuosity). Secondary flow structures are vortical flow patterns that occur in curved arteries due to the combined action of centrifugal forces, adverse pressure gradients and inflow characteristics. Such flow morphologies are greatly affected by pulsatility and multiple harmonics of physiological inflow conditions and vary greatly in size-strength-shape characteristics compared to non-physiological (steady and oscillatory) flows 1 - 7.
Secondary flow structures may ultimately influence the wall shear stress and exposure time of blood-borne particles toward progression of atherosclerosis, restenosis, sensitization of platelets and thrombosis 4 - 6, 8 - 13. Therefore, the ability to detect and characterize these structures under laboratory-controlled conditions is precursor to further clinical investigations.
A common surgical treatment to atherosclerosis is stent implantation, to open up stenosed arteries for unobstructed blood flow. But the concomitant flow perturbations due to stent installations result in multi-scale secondary flow morphologies 4 - 6. Progressively higher order complexities such as asymmetry and loss in coherence can be induced by ensuing stent failures vis-&#224;-vis those under unperturbed flows 5. These stent failures have been classified as &#34;Types I-to-IV&#34; based on failure considerations and clinical severity 14.
This study presents a protocol for the experimental investigation of the complex secondary flow structures due to complete transverse stent fracture and linear displacement of fractured parts (&#34;Type IV&#34;) in a curved artery model. The experimental method involves the implementation of particle image velocimetry (2C-2D PIV) techniques with an archetypal carotid artery inflow waveform, a refractive index matched blood-analog working fluid for phase-averaged measurements 15 - 18. Quantitative identification of secondary flow structures was achieved using concepts of flow physics, critical point theory and a novel wavelet transform algorithm applied to experimental PIV data 5, 6, 19 - 26.

Results presented in Figure 7A-D were generated after post processing secondary flow velocity data (see Figures 5, 6) acquired from 2C-2D PIV system shown in Figure 3A. The inflow condition supplied to the curved artery test section with an idealized &#34;Type IV&#34; stent fracture was the carotid artery waveform shown in Figure 4B. Our previous studies have demonstrated the sensitivity of secondary flow structures to de...

Watch this Scientific Journal Video about Experimental Investigation of Secondary Flow Structures Downstream of a Model Type IV Stent Failure in a 180Ã‚Â° Curved Artery Test Section at JoVE.com

Experimental Investigation of Secondary Flow Structures Downstream of a Model Type IV Stent Failure in a 180&#176; Curved Artery Test Section

Stent implants in stenosed arterial curvatures are prone to "Type IV" failures involving the complete transverse fracture of stents and linear displacement of the fractured parts. We present a protocol for detection of secondary flow (vortical) structures in a curved artery model, downstream of clinically relevant "Type IV" stent failures.

The arterial network in the human vasculature comprises of ubiquitously present blood vessels with complex geometries (branches, curvatures and ...

experimental-investigation-secondary-flow-structures-downstream-model

Research

JoVE Journal

Bioengineering

11.5K Views.  The George Washington University. Stent implants in stenosed arterial curvatures are prone to "Type IV" failures involving the complete transverse fracture of stents and linear displacement of the fractured parts. We present a protocol for detection of secondary flow (vortical) structures in a curved artery model, downstream of clinically relevant "Type IV" stent failures.

Video: Experimental Investigation of Secondary Flow Structures Downstream of a Model Type IV Stent Failure in a 180° Curved Artery Test Section

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well quantifiable fluid shear stresses1.
Our flow chamber design and flow circuit (Fig. 1) contains a transparent viewing region that enables testing of cell adhesion and imaging of cell morphology immediately before flow (Fig. 11A, B), at various time points during flow (Fig. 11C), and after flow (Fig. 11D). These experiments are illustrated with human umbilical cord blood-derived endothelial progenitor cells (EPCs) and porcine EPCs2,3.
This method is also applicable to other adherent cell types, e.g. smooth muscle cells (SMCs) or fibroblasts.
The chamber and all parts of the circuit are easily sterilized with steam autoclaving. In contrast to other chambers, e.g. microfluidic chambers, large numbers of cells (&gt; 1 million depending on cell size) can be recovered after the flow experiment under sterile conditions for cell culture or other experiments, e.g. DNA or RNA extraction, or immunohistochemistry (Fig. 11E), or scanning electron microscopy5. The shear stress can be adjusted by varying the flow rate of the perfusate, the fluid viscosity, or the channel height and width. The latter can reduce fluid volume or cell needs while ensuring that one-dimensional flow is maintained. It is not necessary to measure chamber height between experiments, since the chamber height does not depend on the use of gaskets, which greatly increases the ease of multiple experiments. Furthermore, the circuit design easily enables the collection of perfusate samples for analysis and/or quantification of metabolites secreted by cells under fluid shear stress exposure, e.g. nitric oxide (Fig. 12)6.

We are describing a method to subject adherent cells to laminar flow shear stress in a sterile continuous flow circuit. The cells' adhesion, morphology can be studied through the transparent chamber, samples obtained from the circuit for metabolite analysis and cells harvested after shear exposure for future experiments or culture.

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well ...

Monitoring the Wall Mechanics During Stent Deployment in a Vessel

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the arterial wall lead to tissue injury, which initiates restenosis2-7. This hypothesis needs further investigations including better quantifications of non-uniform strain distribution on the artery following stent implantation. A non-contact surface strain measurement method for the stented artery is presented in this work. ARAMIS stereo optical surface strain measurement system uses two optical high speed cameras to capture the motion of each reference point, and resolve three dimensional strains over the deforming surface8,9. As a mesh stent is deployed into a latex vessel with a random contrasting pattern sprayed or drawn on its outer surface, the surface strain is recorded at every instant of the deformation. The calculated strain distributions can then be used to understand the local lesion response, validate the computational models, and formulate hypotheses for further in vivo study.

Stent-induced arterial strain distributions are characterized using an optical surface strain measurement system. This visualization technique is used to gain insights into the impact of stent implantation on the host vessel.

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the ...

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 ...

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Characterization Of Multi-layered Fish Scales (Atractosteus spatula) Using Nanoindentation, X-ray CT, FTIR, and SEM

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle shells provides unique design principles with potentials for guiding the design of protective materials and systems in the future. Understanding the structure-property relationships for these material systems at the microscale and nanoscale where failure initiates is essential. Currently, experimental techniques such as nanoindentation, X-ray CT, and SEM provide researchers with a way to correlate the mechanical behavior with hierarchical microstructures of these material systems1-6. However, a well-defined standard procedure for specimen preparation of mineralized biomaterials is not currently available. In this study, the methods for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of A. spatula using nanoindentation, FTIR, SEM, with energy-dispersive X-ray (EDX) microanalysis, and X-ray CT are presented.

Characterization Of Multi-layered Fish Scales Atractosteus spatula Using Nanoindentation, X-ray CT, FTIR, and SEM

This paper presents the methods used for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of Atractosteus spatula (A. spatula) using nanoindentation, Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and X-ray computed tomography (X-ray CT). The experimental results have been used to investigate the design principles of protective biological materials.

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle ...

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 ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

Gene Expression Analysis of Endothelial Cells Exposed to Shear Stress Using Multiple Parallel-plate Flow Chambers

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate flow chambers. Endothelial cells form the inner cellular lining of blood vessels and are chronically exposed to the frictional force of blood flow called shear stress. Under physiological conditions, endothelial cells function in the presence of various shear stress conditions. Thus, the application of shear stress conditions in in vitro models can provide greater insight into endothelial responses in vivo. The parallel-plate flow chamber previously published by Lane et al.9 is adapted to study endothelial gene regulation in the presence and absence of steady (non-pulsatile) laminar flow. Key adaptations in the set-up for laminar flow as presented here include a large, dedicated environment to house concurrent flow circuits, the monitoring of flow rates in real-time, and the inclusion of an exogenous reference RNA for the normalization of quantitative real-time PCR data. To assess multiple treatments/conditions with the application of shear stress, multiple flow circuits and pumps are used simultaneously within the same heated and humidified incubator. The flow rate of each flow circuit is measured continuously in real-time to standardize shear stress conditions throughout the experiments. Because these experiments have multiple conditions, we also use an exogenous reference RNA that is spiked-in at the time of RNA extraction for the normalization of RNA extraction and first-strand cDNA synthesis efficiencies. These steps minimize the variability between samples. This strategy is employed in our pipeline for the gene expression analysis with shear stress experiments using the parallel-plate flow chamber, but parts of this strategy, such as the exogenous reference RNA spike-in, can easily and cost-effectively be used for other applications.

Here, a workflow for the culture and gene expression analysis of endothelial cells under fluid shear stress is presented. Included is a physical arrangement for simultaneously housing and monitoring multiple flow chambers in a controlled environment and the use of an exogenous reference RNA for quantitative PCR.

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate ...

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and introduces uncertainty in results due to variability between scorers. To remedy this, automated slide-scanning microscopy as well as conventional flow cytometry methods have been introduced in an attempt to remove scorer bias and improve throughput. However, these methods have their own inherent limitations such as inability to visualize the cytoplasm of the cell and the lack of visual MN verification or image data storage with flow cytometry. Multispectral Imaging Flow Cytometry (MIFC) has the potential to overcome these limitations. MIFC combines the high resolution fluorescent imagery of microscopy with the statistical robustness and speed of conventional flow cytometry. In addition, all collected imagery can be stored in dose-specific files. This paper describes the protocol developed to perform a fully automated version of the MN assay on MIFC. Human lymphoblastoid TK6 cells were enlarged using a hypotonic solution (75 mM KCl), fixed with 4% formalin and the nuclear content was stained with Hoechst 33342. All samples were run in suspension on the MIFC, permitting acquisition of high resolution images of all key events required for the assay (e.g. binucleated cells with and without MN as well as mononucleated and polynucleated cells). Images were automatically identified, categorized and enumerated in the MIFC data analysis software, allowing for automated scoring of both cytotoxicity and genotoxicity. Results demonstrate that using MIFC to perform the in vitro MN assay allows statistically significant increases in MN frequency to be detected at several different levels of cytotoxicity when compared to solvent controls following exposure of TK6 cells to Mitomycin C and Colchicine, and that no significant increases in MN frequency are observed following exposure to Mannitol.

The in vitro micronucleus assay is a well-established method for evaluating genotoxicity and cytotoxicity but scoring the assay using manual microscopy is laborious and suffers from subjectivity and inter-scorer variability. This paper describes the protocol developed to perform a fully automated version of the assay using multispectral imaging flow cytometry.

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and ...