The authors thank Reiner Schultheiss and Wolfgang Schaden for their inspiration for this model. We also thank Christian Dorfm&#252;ller for his all time tremendous efforts to support our research.
Many thanks to Robert G&#246;schl and Hans Hohenegger for careful technical realization of our ideas!

Ethical permission
After obtaining written informed consent of patients, umbilical cords were obtained from Caesarean section at the Department of Gynaecology for isolation of human umbilical vein endothelial cells (HUVECs). Permission was given from the ethics committee of Innsbruck Medical University (no. UN4435).
1. Prepare the IVSWT Water Bath
<ol>
	<li>Prepare 3.5 L&#160;of tap water in an appropriate tank. Water needs to be heated to 37 &#176;C (see Protocol 6).</li>
	<li>Fill the water into the water bath until the water level is approximately 3 cm below the edge. Mind to completely cover the membrane with water. This will require approximately the prepared 3.5 L.</li>
	<li>Connect the temperature sensor to the power supply.</li>
	<li>Put the sensor into the intended fitting on the back wall of the water bath.</li>
	<li>Connect heating to power supply of the temperature sensor. Do not do this without water in the bath as the heater would melt the Plexiglas.</li>
	<li>Wait until the water has reached a stable temperature of 37 &#176;C. Stir the water regularly with some kind of stick to guarantee constant temperature throughout the water bath.</li>
</ol>
2. Prepare Cells and Culture Flasks
<ol>
	<li>Seed cells over night at desired density in T25 culture flasks or culture the cells in the flasks directly until they have reached the desired confluence.</li>
	<li>Before the experiment, align the flasks vertically.</li>
	<li>Fill the culture flasks with culture medium until right below their necks. Do not fill too much medium into the flasks as medium in contact to the neck or the closing cap would get contaminated.</li>
	<li>Screw the flasks with solid caps without filters to prevent contamination from water. Mind that the water in the bath and the bath itself are not sterile.</li>
	<li>Seal the caps with Parafilm before insertion into the water bath.</li>
	<li>Fix sealed culture flasks in the provided stand.</li>
	<li>Insert fixed flask into the water bath. Take care that the middle of the cell culture flask is at the same height as the center of the shock wave applicator&#39;s membrane. A line at the side of the bath guides you.</li>
</ol>
3. Define Treatment Parameters
<ol>
	<li>Define distance between shock wave source and sample. Identify perfect treatment parameters by conducting the parameter finding pilot experiment as described in Figure 6.</li>
	<li>Choose the right treatment parameters (energy flux density, frequency) on the shock wave device. Again, refer to Figure 6.</li>
</ol>
4. Shock Wave Application
<ol>
	<li>Put plenty amounts of commercially available ultrasound transmission gel on the shock wave applicator as well as on the membrane of the water bath. This is to assure coupling. No air or air bubbles should be between the applicator and the membrane as it would absorb shock waves.</li>
	<li>Connect applicator to the membrane and hold it stable in the center of the membrane. Take care that it is aligned horizontally.</li>
	<li>Make sure that the correct vertical position of the probe inside the water bath is in line with the indicated marking at the side of the bath.</li>
	<li>Hold centers of the applicator and the flask stable in a horizontal line.</li>
	<li>Activate the shock wave device and apply the impulses while keeping the culture flask as well as the applicator in a stable position during the whole procedure.</li>
</ol>
5. After Treatment
<ol>
	<li>Take the culture flask out of the water bath.</li>
	<li>Dry it with common paper towels.</li>
	<li>Wipe the flask accurately with disinfecting agents.</li>
	<li>Remove Parafilm sealing and cap.</li>
	<li>Pipette the Medium inside the flasks into centrifugal tubes.</li>
	<li>Centrifuge appropriately. Centrifuging parameters depend on the used cell type, e.g. for HUVECs, use 290 g at room temperature.</li>
	<li>Add 5ml of cell culture medium to the flask. Resuspend the centrifuged cell pellet with 2 ml of cell culture medium. Pipette the suspension into the flask. Thus, cell fragments or cells that may have been detached during treatment do not get lost.</li>
</ol>
Pitfalls
<ol>
	<li>Preparation as well as treatment should be carried out under sterile conditions inside laminar flow to avoid contamination of the cell culture.</li>
	<li>Good cell culture practice is recommended to avoid contamination of the probes. In particular disinfection of the outside of the flasks is strongly recommended before putting them back to the incubator. The water bath, as well as the water inside, is not sterile!</li>
	<li>Do not connect the heater to the power supply unless the water bath is filled with water to avoid damage to the Plexiglas built bath.</li>
	<li>Use generous amounts of ultrasound gel on the shock wave applicator to guarantee proper coupling and wave propagation into the bath. Air and even small air bubbles do absorb shock waves!</li>
	<li>Check position of the probes inside the water bath in relation to the applicator to ensure treatment of the entire growth area.</li>
	<li>Do not dunk probes too deep into the water to avoid contamination.</li>
</ol>

<ol>
	<li>Haupt, G., Haupt, A., Ekkernkamp, A., Gerety, B., Chvapil, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+shock+waves+on+fracture+healing.">Influence of shock waves on fracture healing.</a> Urology. 39, 529-532 (1992).</li><li>Schaden, W., Fischer, A., Sailler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracorporeal+shock+wave+therapy+of+nonunion+or+delayed+osseous+union.">Extracorporeal shock wave therapy of nonunion or delayed osseous union.</a> Clin. Orthop. Relat. Res. 387, 90-94 (2001).</li><li>Schaden, W., 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=Shock+wave+therapy+for+acute+and+chronic+soft+tissue+wounds:+a+feasibility+study.">Shock wave therapy for acute and chronic soft tissue wounds: a feasibility study.</a> J. Surg. Res. 143, 1-12 (2007).</li><li>Tepek&#246;yl&#252;, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shock+wave+treatment+induces+angiogenesis+and+mobilizes+endogenous+CD31/CD34-positive+endothelial+cells+in+a+hindlimb+ischemia+model:+Implications+for+angiogenesis+and+vasculogenesis.">Shock wave treatment induces angiogenesis and mobilizes endogenous CD31/CD34-positive endothelial cells in a hindlimb ischemia model: Implications for angiogenesis and vasculogenesis.</a> J. Thorac. Cardiovasc. Surg. 146, 971-978 (2013).</li><li>Nishida, T., 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=Extracorporeal+cardiac+shock+wave+therapy+markedly+ameliorates+ischemia-induced+myocardial+dysfunction+in+pigs+in+vivo.">Extracorporeal cardiac shock wave therapy markedly ameliorates ischemia-induced myocardial dysfunction in pigs in vivo.</a> Circulation. 110, 3055-3061 (2004).</li><li>Fukumoto, Y., 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=Extracorporeal+cardiac+shock+wave+therapy+ameliorates+myocardial+ischemia+in+patients+with+severe+coronary+artery+disease.">Extracorporeal cardiac shock wave therapy ameliorates myocardial ischemia in patients with severe coronary artery disease.</a> Coron. Artery Dis. 17, 63-70 (2006).</li><li>Gotte, G., Amelio, E., Russo, S., Marlinghaus, E., Musci, G., Suzuki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-time+non-enzymatic+nitric+oxide+synthesis+from+L-arginine+and+hydrogen+peroxide+induced+by+shock+waves+treatment.">Short-time non-enzymatic nitric oxide synthesis from L-arginine and hydrogen peroxide induced by shock waves treatment.</a> FEBS Lett. 520, 153-155 (2002).</li><li>Wang, F. S., Wang, C. J., Huang, H. J., Chung, H., Chen, R. F., Yang, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+shock+wave+mediates+membrane+hyperpolarization+and+Ras+activation+for+osteogenesis+in+human+bone+marrow+stromal+cells.">Physical shock wave mediates membrane hyperpolarization and Ras activation for osteogenesis in human bone marrow stromal cells.</a> Biochem. Biophys. Res. Commun. 287, 648-655 (2001).</li><li>Mittermayr, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracorporeal+shock+wave+therapy+(ESWT)+minimizes+ischemic+tissue+necrosis+irrespective+of+application+time+and+promotes+tissue+revascularization+by+stimulating+angiogenesis.">Extracorporeal shock wave therapy (ESWT) minimizes ischemic tissue necrosis irrespective of application time and promotes tissue revascularization by stimulating angiogenesis.</a> Ann. Surg. 253, 1024-1032 (2011).</li><li>Baker, 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=Use+of+the+mouse+aortic+ring+assay+to+study+angiogenesis.">Use of the mouse aortic ring assay to study angiogenesis.</a> Nat. Protoc. 22, 89-104 (2011).</li></ol>

The authors have nothing to disclose.

The significance of the proposed model for in vitro shock wave treatment is the fact that waves can propagate after passing the cell culture in contrast to existing models. Thereby, disturbing physical effects such as tensile forces can be avoided. The model more closely resembles in vivo conditions than that by others applying waves to their cell culture flasks directly.
An additional advantage is the possibility of varying the distance between shock wave source and cells. T...

Shock waves are sound pressure waves arising from a sudden release of energy, e.g. as thunder when lightning. In medicine shock waves have been used for over 30 years&#160;in Lithotripsy for the disintegration of kidney stones. Since the incidental finding of iliac bone thickening in Lithotripsy patients in the early 1980s, first studies were conducted to evaluate the effect of shock wave treatment (SWT) on bone healing1. Impressive results of improved healing of long-bone nonunions could be observed2. Subsequently, indications were expanded to soft tissue wounds3. Basic research findings showed that shock waves do cause a biological stimulus to the target tissue without any subsequent damage. Release of angiogenic growth factors (e.g. VEGF, PlGF, FGF) is followed by significant angiogenesis. This led to a further expansion of indications towards ischemic pathologies. Our group and others showed the positive effect of SWT on ischemic heart disease in animal models as well as in clinical trials4-6.
However, the exact mechanism of how the physical stimulus of SWT is translated into a biological signal (mechanotransduction) remains largely unknown. As the interest in SWT from several fields of medicine increases continuously, the quest for the mechanism is getting more and more intense. Therefore, in&#160;vitro shock wave experiments are gaining importance. Besides reduction of animal experiments and cost-effectiveness, the biggest advantage of in&#160;vitro shock wave treatment (IVSWT) may be the possibility of studying the specific behavior of a certain cell type. In shock wave mediated tissue regeneration most likely all cells of the treated tissue are involved, even systemic effects are discussed. Nevertheless, each cell type plays a specific role and has its own intrinsic function. IVSWT enables us to detect this particular function and thereby gives us better understanding of the complex underlying processes.
Today&#39;s knowledge about shock wave&#39;s effects on cell cultures includes the increase of proliferation, alteration of cell membrane receptors, increase and acceleration of cell differentiation, release of growth factors and chemo-attractants as well as increased cell migration7-9.
Distracting physical effects in most in&#160;vitro models Various methods of applying shock waves onto cell cultures have been described. This fact leads to the problem that it is highly difficult to compare results, as physical conditions of cell stimulation are quite different between these models. In general, all existing models focus on how to best apply shock waves onto cells.
However, this question remains: What happens to the waves after passing the cell culture? The main problem is that the difference of the acoustic impedance of the cell culture medium and the ambient air is that high, that more than 99% of shock waves get reflected Figure 1.
Due to the difference in acoustic impedance of the two media the waves are not only reflected but a phase-shift of 180&#176; occurs resulting in strong tensile forces to the cells Figure 2.
Acoustic impedance is defined as the product of the density of a material and its sound velocity Z=&#961; x c. For water the acoustic impedance is ZWater= 1,440,000 Ns/m3, for air it is only 420 Ns/m3. The large difference of these two values results in reflection and phase shift of shock waves. The phase shift turns a positive pressure pulse into a tensile wave.
Even if this tensile force is not harmful to the cells, it interferes with the idea of mimicking in vivo shock wave effects in vitro. In vivo these tensile forces hardly occur due to large body structures.
Furthermore, the back running waves can even disturb the incoming ones. This may cause interference. Two types of interference are known. Constructive interference means that both waves are added thereby resulting in doubled amplitude&#160;Figure 3. Destructive interference occurs if waves meet diametrically opposed. It causes abolishment of waves (Figure 3). Therefore, IVSWT needs a model that enables shock waves to propagate after passing the cell culture.
IVSWT water bath
Considerations following the above mentioned concerns lead us to design a water bath for avoiding the described problems Figure 4. Basically, it consists of a Plexiglas built container with a membrane to connect every kind of shock wave applicator. For coupling between this membrane and the applicator ultrasound transmission gel has to be used. The water bath is filled with degassed water to avoid cavitation that would occur if gas was soluted within the water. A heater at the bottom with a temperature sensor connected to a control unit enables to regulate temperature for imitation of in vivo conditions and to avoid cell cultures to cool down during the procedure. Temperature can be held stable at 37 degrees centigrade as it is done in an incubator. A holder for the cell samples allows for immersing any kind of culture flask or tube. Thereby, the sample vessel needs to be completely filled with culture medium, as air bubbles would block shock waves! A wedge shaped absorber at the back wall of the bath destructs waves in order not to get reflected and run back to avoid interference.
A further advantage to other IVSWT models is the possibility of varying the distance between the applicator and the culture flasks. Findings of our group and others who use this model clearly show that every cell type reacts very specifically to different treatment parameters. Moreover, defining the distance between the source of the waves and the sample is crucial as it enables us to control the cells to be at a specific position in relation to the focus of the shock wave applicator.

<table border="0" cellpadding="0" cellspacing="0" width="2002">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:420px;">Orthogold shock wave device</td>
			<td colspan="2" style="width:756px;">Tissue Regeneration Technologies, Woodstock, GA &#8211; manufactured by MTS-Europe GmbH, Konstanz, Germany</td>
			<td style="width:827px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:420px;">IVSWT Water Bath V2.0</td>
			<td style="width:537px;">Johann Hohenegger - Technical Products</td>
			<td style="width:219px;">-</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">EBM-2 Basal Medium 500 m +EGM-2 SingleQuot Suppl.&#38;Growth Factors</td>
			<td style="width:537px;">Lonza</td>
			<td style="width:219px;">CC-3156 &#38; CC-4176</td>
			<td>This medium was used for the shown experiments with HUVECs to fill the cell culture flask. For other cell types, use the recommended medium.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"><a href="http://www.labplanet.com/pechiney-parafilm-m-pm996.html">Pechiney&#160;Parafilm M PM996</a></td>
			<td style="width:537px;">Pechiney Plastic Packaging</td>
			<td>PH-LF-PM996-EA at labplanet.com</td>
			<td>for sealing flasks</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:420px;">Falcon Serological pipets 25ml</td>
			<td style="width:537px;">Becton Dickinson Labware</td>
			<td style="width:219px;">357525</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:420px;">CellMate II Serological Pipette&#160;</td>
			<td style="width:537px;">Matrix Technologies</td>
			<td style="width:219px;">-</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:420px;">Skintact Ultrasonic Gel</td>
			<td style="width:537px;">Skintact</td>
			<td style="width:219px;">UL-01 250 ml</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:420px;">T25 cell culture flasks</td>
			<td style="width:537px;">COSTAR</td>
			<td style="width:219px;">3056</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:420px;">mikrozid disinfectant</td>
			<td style="width:537px;">Sch&#252;lke</td>
			<td style="width:219px;">-</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:420px;">3,5l degassed water</td>
			<td style="width:537px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:420px;">paper towels</td>
			<td style="width:537px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

shock wave application to cell cultures

Shock waves nowadays are well known for their regenerative effects. Basic research findings showed that shock waves do cause a biological stimulus to target cells or tissue without any subsequent damage. Therefore, in vitro experiments are of increasing interest. Various methods of applying shock waves onto cell cultures have been described. In general, all existing models focus on how to best apply shock waves onto cells.
However, this question remains: What happens to the waves after passing the cell culture? The difference of the acoustic impedance of the cell culture medium and the ambient air is that high, that more than 99% of shock waves get reflected! We therefore developed a model that mainly consists of a Plexiglas built container that allows the waves to propagate in water after passing the cell culture. This avoids cavitation effects as well as reflection of the waves that would otherwise disturb upcoming ones. With this model we are able to mimic in vivo conditions and thereby gain more and more knowledge about how the physical stimulus of shock waves gets translated into a biological cell signal (&#8220;mechanotransduction&#34;).

Using the described method we applied shock waves to human umbilical vein endothelial cells (HUVECs) that we afore isolated from umbilical cords. Umbilical cords were obtained from elective caesarian sections.
HUVECs were treated at a confluence of 90% in a T25 cell culture flask with an electrohydraulic shock wave therapy system. Treatment parameters were an energy flux density of 0.1 mJ/mm2 and a frequency of 5 Hz. 300 impulses were applied from a distance of 5 cm from the shock w...

Watch this Scientific Journal Video about Shock Wave Application to Cell Cultures at JoVE.com

Shock Wave Application to Cell Cultures

Shock waves nowadays are well known for their regenerative effects. Therefore in vitro experiments are of increasing interest. We therefore developed a model for in vitro shock wave trials (IVSWT) that enables us to mimic in vivo conditions thereby avoiding distracting physical effects.

Shock waves nowadays are well known for their regenerative effects. Basic research findings showed that shock waves do cause a biological stimulus to ...

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SCIENTISTS IN ACTION

12.7K ビュー.  Innsbruck Medical University. 衝撃波は、今日ではその再生効果によく知られている。従ってインビトロ実験は、関心の高まりである。そこで我々は、それによって気が散るの物理的効果を回避すること、生体内の状態に模倣することを可能にin vitroでの衝撃波試験（IVSWT）のためのモデルを開発しました。 

ビデオ: Shock Wave Application to Cell Cultures

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

High-throughput, Automated Extraction of DNA and RNA from Clinical Samples using TruTip Technology on Common Liquid Handling Robots

TruTip is a simple nucleic acid extraction technology whereby a porous, monolithic binding matrix is inserted into a pipette tip. The geometry of the monolith can be adapted for specific pipette tips ranging in volume from 1.0 to 5.0 ml. The large porosity of the monolith enables viscous or complex samples to readily pass through it with minimal fluidic backpressure. Bi-directional flow maximizes residence time between the monolith and sample, and enables large sample volumes to be processed within a single TruTip. The fundamental steps, irrespective of sample volume or TruTip geometry, include cell lysis, nucleic acid binding to the inner pores of the TruTip monolith, washing away unbound sample components and lysis buffers, and eluting purified and concentrated nucleic acids into an appropriate buffer. The attributes and adaptability of TruTip are demonstrated in three automated clinical sample processing protocols using an Eppendorf epMotion 5070, Hamilton STAR and STARplus liquid handling robots, including RNA isolation from nasopharyngeal aspirate, genomic DNA isolation from whole blood, and fetal DNA extraction and enrichment from large volumes of maternal plasma (respectively).

TruTip is a simple nucleic acid extraction technology whereby a porous, monolithic binding matrix is inserted into a pipette tip. Consequently, the sample preparation format is compatible with most liquid handling instruments, and can be used for many medium to high-throughput clinical applications and sample types.

TruTip is a simple nucleic acid extraction  technology whereby a porous, monolithic binding matrix is inserted into a  pipette tip. The geometry of the ...

Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim to use these principles to create tissues with predefined collagen architectures. A full understanding of the exact underlying processes of collagen remodeling to control the final tissue architecture, however, is lacking. In particular, little is known about the (re)orientation of collagen fibers in response to changes in tissue mechanical loading conditions. We developed an in&#160;vitro model system, consisting of biaxially-constrained myofibroblast-seeded fibrin constructs, to further elucidate collagen (re)orientation in response to i) reverting biaxial to uniaxial static loading conditions and ii) cyclic uniaxial loading of the biaxially-constrained constructs before and after a change in loading direction, with use of the Flexcell FX4000T loading device. Time-lapse confocal imaging is used to visualize collagen (re)orientation in a nondestructive manner.
Cell and collagen organization in the constructs can be visualized in real-time, and an internal reference system allows us to relocate cells and collagen structures for time-lapse analysis. Various aspects of the model system can be adjusted, like cell source or use of healthy and diseased cells. Additives can be used to further elucidate mechanisms underlying collagen remodeling, by for example adding MMPs or blocking integrins. Shape and size of the construct can be easily adapted to specific needs, resulting in a highly tunable model system to study cell and collagen (re)organization.

This model system starts from a myofibroblast-populated fibrin gel that can be used to study endogenous collagen (re)organization real-time in a nondestructive manner. The model system is very tunable, as it can be used with different cell sources, medium additives, and can be adapted easily to specific needs.

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim ...

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 ...

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards vascular disease pathologies. Unhealthy arteries often present with wall stiffening, scarring, or partial stenosis which may all affect fluid flow rates, and the magnitude of pulsatile flow, or pulsatility index. Replication of various flow conditions is the result of tuning a flow pressure damping chamber downstream of a blood pump. Introduction of air within a closed flow system allows for a compressible medium to absorb pulsatile pressure from the pump, and therefore vary the pulsatility index. The method described herein is simply reproduced, with highly controllable input, and easily measurable results. Some limitations are recreation of the complex physiological pulse waveform, which is only approximated by the system. Endothelial cells, smooth muscle cells, and fibroblasts are affected by the blood flow through the artery. The dynamic component of blood flow is determined by the cardiac output and arterial wall compliance. Vascular cell mechano-transduction of flow dynamics may trigger cytokine release and cross-talk between cell types within the artery. Co-culture of vascular cells is a more accurate picture reflecting cell-cell interaction on the blood vessel wall and vascular response to mechanical signaling. Contribution of flow dynamics, including the cell response to the dynamic and mean (or steady) components of flow, is therefore an important metric in determining disease pathology and treatment efficacy. Through introducing an in vitro co-culture model and pressure damping downstream of blood pump which produces simulated cardiac output, various arterial disease pathologies may be investigated.

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

This protocol replicates physiological or pathological blood flow in vitro to aid in determining cell response in disease pathologies. By introducing a pressure damping chamber downstream of a blood pump, blood flow across the vasculature can be recapitulated and imposed on a monolayer of vascular endothelium or a mimetic co-culture.

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards ...

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...

Large-area Scanning Probe Nanolithography Facilitated by Automated Alignment and Its Application to Substrate Fabrication for Cell Culture Studies

Scanning probe microscopy has enabled the creation of a variety of methods for the constructive (&#39;additive&#39;) top-down fabrication of nanometer-scale features. Historically, a major drawback of scanning probe lithography has been the intrinsically low throughput of single probe systems. This has been tackled by the use of arrays of multiple probes to enable increased nanolithography throughput. In order to implement such parallelized nanolithography, the accurate alignment of probe arrays with the substrate surface is vital, so that all probes make contact with the surface simultaneously when lithographic patterning begins. This protocol describes the utilization of polymer pen lithography to produce nanometer-scale features over centimeter-sized areas, facilitated by the use of an algorithm for the rapid, accurate, and automated alignment of probe arrays. Here, nanolithography of thiols on gold substrates demonstrates the generation of features with high uniformity. These patterns are then functionalized with fibronectin for use in the context of surface-directed cell morphology studies.

Here we present a protocol for wide-area scanning probe nanolithography enabled by the iterative alignment of probe arrays, as well as the utilization of lithographic patterns for cell-surface interaction studies.

Scanning probe microscopy has enabled the creation of a variety of methods for the constructive (&#39;additive&#39;) top-down fabrication of ...

Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases including cancer. However, it is challenging to clarify the complex mechanism of intercellular interactions in highly heterogeneous cell populations using conventional co-culture systems because the heterogeneity of the cell subpopulation is obscured by the average values; the conventional co-culture systems can only be used to describe the population signal, but are incapable of tracking individual cells behavior. Furthermore, conventional single-cell experimental methods have low efficiency in cell manipulation because of the Poisson distribution. Microfabricated devices are an emerging technology for single-cell studies because they can accurately manipulate single cells at high-throughput and can reduce sample and reagent consumption. Here, we describe the concept and application of a microfluidic chip for multiple single-cell co-cultures. The chip can efficiently capture multiple types of single cells in a culture chamber (~46%) and has a sufficient culture space useful to study the cells&#39; behavior (e.g., migration, proliferation, etc.) under cell-cell interaction at the single-cell level. Lymphatic endothelial cells and oral squamous cell carcinoma were used to perform a single-cell co-culture experiment on the microfluidic platform for live multiple single-cell interaction studies.

This report describes a microfluidic chip-based method to set up a single cell culture experiment in which high-efficiency pairing and microscopic analysis of multiple single cells can be achieved.

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases ...

Increasing Durability of Dissociated Neural Cell Cultures Using Biologically Active Coralline Matrix

Cultures of dissociated hippocampal neuronal and glial cells are a valuable experimental model for studying neural growth and function by providing high cell isolation and a controlled environment. However, the survival of hippocampal cells in vitro is compromised: most cells die during the first week of culture. It is therefore of great importance to identify ways to increase the durability of neural cells in culture.
Calcium carbonate in the form of crystalline aragonite derived from the skeleton of corals can be used as a superior, active matrix for neural cultures. By nurturing, protecting, and activating glial cells, the coral skeleton enhances the survival and growth of these cells in vitro better than other matrices.
This protocol describes a method for cultivating hippocampal cells on a coralline matrix. This matrix is generated by attaching grains of coral skeletons to culture dishes, flasks, and glass coverslips. The grains assist in improving the environment of the cells by introducing them to a fine three-dimensional (3D) environment to grow on and to form tissue-like structures. The 3D environment introduced by the coral skeleton can be optimized for the cells by grinding, which enables control over the size and density of the grains (i.e., the matrix roughness), a property that has been found to influence glial cells activity. Moreover, the use of grains makes the observation and analysis of the cultures easier, especially when using light microscopy. Hence, the protocol includes procedures for generation and optimization of the coralline matrix as a tool to improve the maintenance and functionality of neural cells in vitro.

Dissociated hippocampal cell culture is a pivotal experimental tool in neuroscience. Neural cell survival and function in culture is enhanced when coralline skeletons are used as matrices, due to their neuroprotective and neuromodulative roles. Hence, neural cells grown on coralline matrix show higher durability, and thereby are more adequate for culturing.

Cultures of dissociated hippocampal neuronal and glial cells are a valuable experimental model for studying neural growth and function by providing high ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...