Name: An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues (Video) | JoVE Journal
Uploaded: 2019-01-07T14:00:00.000+00:00
Duration: 7 min 50 s
Description: Learn an in vitro flow chamber model to study bacterial adherence to graft tissues for infective endocarditis research. This method reveals molecular interactions driving tissue infectiveness under standardized conditions.

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5 minutes

This study was sponsored by a grant of the Research Fund KU Leuven (OT/14/097) given to RH. TRV was Postdoctoral Fellow of the FWO Research Foundation - Flanders (Belgium; Grant Number - 12K0916N) and RH is supported by the Clinical Research Fund of UZ Leuven.

1. Preparing Graft Tissues for In Vitro Studies
Note: Three types of tissues were used: Bovine Pericardium patch (BP), Cryopreserved Homograft (CH) and Bovine Jugular Vein grafts (BJV). In case of BJV conduit and CH (tissue processed by the European Homograft Bank (EHB) and stored in liquid nitrogen prior to use), both the wall and valvular leaflets were used. BP patch and BJV conduit were purchased from the manufacturers. Prior to use, thaw the CH following the EHB instructions12.
<ol>
	<li>Rinse all tissues with 0.9% NaCl prior to use.</li>
	<li>Prepare tissue biopsies using a disposable skin biopsy punch to cut circular tissue pieces (10 mm in diameter).</li>
	<li>Cut all tissue patches to the same height using disposable sterile scalpels.</li>
	<li>For tissues fixed with glutaraldehyde (for example BJV conduit), incubate graft pieces overnight at 4 &#176;C with 200 g/L of human albumin to neutralize the fixative.</li>
	<li>Wash out residues of glutaraldehyde with 0.9% NaCl in a microtiter plate.&#160; Repeat 3 times for 1 minute.</li>
</ol>
2. Preparing Bacteria for Perfusion Experiments
Note: Three bacterial isolates were used: S. aureus Cowan (ATCC 12598), S. epidermidis ATCC 149900 and S. sanguinis NCTC 7864. S. aureus and S. epidermidis were grown at 37 &#176;C in tryptic soy broth (TSB) and S. sanguinis was grown at 37 &#176;C with 5% CO2 in brain heart infusion broth (BHI).
<ol>
	<li>Prepare overnight culture of bacteria on a solid blood agar plate.
	<ol>
		<li>Use a sterile loop to scrape the frozen bacteria off and inoculate onto a Mueller-Hinton blood agar plate for overnight culture at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Use a sterile inoculation loop to pick up a single colony from the overnight blood agar culture and inoculate into 10 mL of TSB or BHI in a 14&#160;mL tube and culture overnight at 37 &#176;C.</li>
	<li>Centrifuge overnight cultures (3000 x g, 4 &#176;C, 10 min) and resuspend the pellets in 10 mL of phosphate buffered saline (PBS). Place 14&#160;mL tubes on ice.</li>
	<li>Prepare an aliquot of 3.7 mg/mL solution of 5(6)-carboxy-fluorescein N-hydroxy-succinimidyl ester (CF) in ethanol and store at -20 &#176;C. Further dilute the stock of CF to 150 &#181;g/mL using &#39;ultrapure&#39; water. 
	Note: Protect tubes from light using aluminum foil and store at -20 &#176;C.</li>
	<li>Centrifuge the bacteria (3000 x g, 4 &#176;C, 10 min) and resuspend the pellets in 800 &#181;L of PBS and add 200 &#181;L of the 150 &#181;g/mL CF solution (final concentration of 30 &#181;g/mL used for perfusion experiments). Protect tubes from light with aluminum foil and incubate for 30 min using an orbital shaker.</li>
	<li>After labeling, block with 2% of bovine serum albumin (BSA) solution in PBS and spin (3000 x g, 4 &#176;C, 10 min). Follow with a wash step using 10 mL of PBS and pellet bacteria by centrifugation (3000 x g, 4 &#176;C, 10 min).</li>
	<li>Dilute bacteria with PBS to obtain 107 colony-forming units (CFU)/mL (verified by CFU counting on Mueller-Hinton blood agar plates), which corresponds to an OD600 (optical density) of 0.65. Keep the tubes in the dark on ice prior to perfusion experiments. 
	Note. Keep in mind that OD600 measurements reflect the approximate number of bacteria. To count the effective inoculation dose, the serial dilution method is an additional necessary step to verify the OD based numbers as described in section 3.8.</li>
</ol>
3. In vitro Perfusion Experiments using a Parallel-Plate Flow Chamber
<ol>
	<li>Mount tissue biopsies of 10 mm in diameter and the same thickness (prepared in the steps 1.1 - 1.5) into a flow chamber system with the inner surface facing up to get in contact with the bacterial suspension. 
	Note: The same tissue thickness across various grafts ensures that the same tissue height is reached in the channel allowing laminar flow in all conditions. All elements of the flow chamber are presented and described in Figure 1.
	<ol>
		<li>To begin the protocol, place the round tissue piece between a microscope slide with an 8 mm circular perforation and a rubber gasket. 
		Note: The microscope slide possesses the ultra-thin bottom film to allow the generation of the 8 mm hole. Together with the rubber sheet, it fixes the tissue to enable the direct contact between the specimen and the flowing medium and also prevents the dislocation of the biopsy during the experiment. The surface of the investigated tissue, which is exposed to the flow (smaller diameter) cannot be manipulated by the forceps.</li>
		<li>Insert the holder with the tissue into the gasket sheet that is embedded in the bottom metal frame of the chamber.&#160;</li>
		<li>Attach the upper metal frame with the corresponding gasket sheet onto the bottom part of the chamber with the previously inserted tissue holder. Subsequently mount the entire chamber with eight screws and screw nuts. Make sure that the chamber height is always the same across grafts. 
		Note: The chamber height should be determined always upon tightening the screws. Use a caliper or ruler.</li>
	</ol>
	</li>
	<li>Connect the flow chamber with a peristaltic pump and the fluid reservoir with the tubes.</li>
	<li>Perfuse the tissues with suspensions of 107 CFU/mL (verified by CFU counting and related to OD600 measurements) fluorescently-labeled bacteria in PBS with a shear stress of 3 dyne/cm2 (dyne per square centimeter pressure unit) by means of the peristaltic pump (flow rate 4 mL/min) for 1 h using a 400 mL bacterial reservoir (In-house design, Figure 1) conditioned at 37 &#176;C using a plate thermostat (Table of Materials).</li>
	<li>Recirculate continuously the 100 mL bacterial suspension&#160;using the same&#160;collection reservoir.</li>
	<li>After perfusion, dismantle the chamber to release the graft and wash the tissue piece two times&#160;with 4&#160;mL of PBS&#160;in a 12-well plate using the laboratory orbital shaker for 3 min each. Subsequently cut the inner part of the graft using a skin biopsy punch of a smaller diameter.</li>
	<li>Place each tissue biopsy into a separate 14&#160;mL tube containing 1 mL of sterile 0.9% NaCl. Label the tube as #1.</li>
	<li>Detach the bacteria from the tissue using a sonication bath for 10 min (amplitude = 100% and frequency = 45 kHz). 
	Note: Full detachment of bacteria from the tissue grafts should be evaluated upon incubation of patches overnight at 37 &#176;C in TSB liquid medium followed by OD600 measurements compared to control patches treated with a bacteria free solution.</li>
	<li>Use a serial dilution method on Mueller-Hinton blood agar plates to count CFUs.
	<ol>
		<li>Prepare a single 14&#160;mL tube with 10 mL of sterile saline to make&#160;serial dilutions of the bacterial suspension obtained after sonication. Label this tube as #2. 
		Note: For each tissue experiment one tube with 10 mL of 0.9% NaCl is necessary.</li>
		<li>Prepare three 14&#160;mL tubes with 10 mL of sterile 0.9% NaCl for serial dilutions of initial bacterial suspension from step 2.7. Label the tubes as follows #3, #4, #5. 
		Note: This step is necessary to know the real CFU number in bacterial suspension used for the perfusion experiment.</li>
		<li>Vortex mix each tube for 15 s. Vortex the tubes with the tissue biopsy as well as the initial&#160;bacterial suspension to make serial dilutions.</li>
		<li>Prepare three agar plates, two for the tissue experiment (perfusion of bacteria and control perfusion of PBS) and the third one for the initial bacterial suspension used for perfusions.</li>
		<li>Label three sectors per plate for the tissue experiment in the following manner 10-1, 10-3 and 10-4. To count the number of CFUs in the&#160;bacterial perfusates, label the&#160;plate as follows: 10-1, 10-3, 10-5 and 10-7. 
		Note: All indications on agar plates such as 10-1, 10-3 and so on refer to the final number of CFU/mL calculated on the next day. Control plate does not require any sectors. Before use, blood agar plates should be placed under the laminar hood and opened to remove excess moisture.</li>
		<li>To continue preparing the&#160;serial dilutions, transfer 100 &#181;L of tube #1 to tube #2 and mix vigorously with vortex.</li>
		<li>Spread 100 &#181;L of the contents of tube #1 and #2 onto the corresponding sectors 10-1 and 10-3 of the agar plate. Likewise, spread 10 &#181;L of tube #2 on the sector 10-4, repeat this step 4 times to obtain 4 separate growths from each volume of 10 &#181;L. 
		Note: Due to the small volume used for plating onto the sector 10-4, it is advised to have multiple number of droplets to make an average number of grown CFUs.</li>
		<li>To prepare the serial dilutions of the initial&#160;culture, transfer 100 &#181;L of bacterial suspension from step 2.7 to tube #3 and mix vigorously with vortex. Add 100 &#181;L of tube #3 to tube #4 and mix well, repeat the procedure for subsequent tube #5.</li>
		<li>Spread 100 &#181;L of the contents of tubes #3, #4, #5 and the adjusted bacterial suspension (step 2.7), respectively, onto sectors 10-3, 10-5, 10-7 and 10-1 of the blood agar plate.</li>
		<li>Leave the blood agar plates in the laminar hood to air dry the bacterial spreads, typically for 10 minutes. Afterwards, place the plates at 37 &#176;C for overnight incubation.</li>
		<li>After overnight incubation, count the bacterial colonies to obtain the number of CFUs resulting from the adhesion to the tissue biopsies as well as&#160;CFUs/mL in the starting bacterial suspension used for the perfusion. Express results as CFU/cm2.</li>
	</ol>
	</li>
</ol>
4. Fluorescence Microscopy of Adhered Bacteria to Graft Tissues upon Perfusion
<ol>
	<li>After perfusion, wash tissue pieces with PBS (see step 3.5) and cut the inner part of a graft using a punch of a smaller diameter.</li>
	<li>Prepare a 6-well plate and place droplets of mounting medium (Table of Materials).</li>
	<li>Place each piece of tissue with its perfused surface downward on a single drop of mounting medium.</li>
	<li>Read a plate using a fluorescence scanner (Table of Materials). Set parameters of excitation and emission wavelengths according to a fluorophore used for bacterial labeling.</li>
</ol>

<ol><li>Que, Y. A., Moreillon, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Infective+endocarditis%5BTitle%5D)+AND+%22Nature+Reviews+Cardiology%22%5BJournal%5D)">Infective endocarditis.</a> Nature Reviews Cardiology. 8 (6), 322-336 (2011).</li>
<li>Werdan, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanisms+of+infective+endocarditis%3A+pathogen-host+interaction+and+risk+states%5BTitle%5D)+AND+%22Nature+Reviews+Cardiology%22%5BJournal%5D)">Mechanisms of infective endocarditis: pathogen-host interaction and risk states.</a> Nature Reviews Cardiology. 11 (1), 35-50 (2014).</li>
<li>Moreillon, P., Que, Y. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Infective+endocarditis%5BTitle%5D)+AND+%22The+Lancet%22%5BJournal%5D)">Infective endocarditis.</a> The Lancet. 363 (9403), 139-149 (2004).</li>
<li>Jalal, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Selective+propensity+of+bovine+jugular+vein+material+to+bacterial+adhesions%3A+An+in+vitro+study%5BTitle%5D)+AND+%22International+Journal+of+Cardiology%22%5BJournal%5D)">Selective propensity of bovine jugular vein material to bacterial adhesions: An in vitro study.</a> International Journal of Cardiology. 198, 201-205 (2015).</li>
<li>Sharma, A., Cote, A. T., Hosking, M. C. K., Harris, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Systematic+Review+of+Infective+Endocarditis+in+Patients+With+Bovine+Jugular+Vein+Valves+Compared+With+Other+Valve+Types%5BTitle%5D)+AND+%22JACC+Cardiovascular+Interventions%22%5BJournal%5D)">A Systematic Review of Infective Endocarditis in Patients With Bovine Jugular Vein Valves Compared With Other Valve Types.</a> JACC Cardiovascular Interventions. 10 (14), 1449-1458 (2017).</li>
<li>Malekzadeh-Milani, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Incidence+and+predictors+of+Melody%28R%29+valve+endocarditis%3A+a+prospective+study%5BTitle%5D)+AND+%22Archives+of+Cardiovascular+Diseases%22%5BJournal%5D)">Incidence and predictors of Melody(R) valve endocarditis: a prospective study.</a> Archives of Cardiovascular Diseases. 108 (2), 97-106 (2015).</li>
<li>Hill, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Management+of+prosthetic+valve+infective+endocarditis%5BTitle%5D)+AND+%22American+Journal+of+Cardiology%22%5BJournal%5D)">Management of prosthetic valve infective endocarditis.</a> American Journal of Cardiology. 101 (8), 1174-1178 (2008).</li>
<li>Claes, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clumping+factor+A%2C+von+Willebrand+factor-binding+protein+and+von+Willebrand+factor+anchor+Staphylococcus+aureus+to+the+vessel+wall%5BTitle%5D)+AND+%22Journal+of+Thrombosis+and+Haemostasis%22%5BJournal%5D)">Clumping factor A, von Willebrand factor-binding protein and von Willebrand factor anchor Staphylococcus aureus to the vessel wall.</a> Journal of Thrombosis and Haemostasis. 15 (5), 1009-1019 (2017).</li>
<li>Fowler, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cellular+invasion+by+Staphylococcus+aureus+involves+a+fibronectin+bridge+between+the+bacterial+fibronectin-binding+MSCRAMMs+and+host+cell+beta1+integrins%5BTitle%5D)+AND+%22European+Journal+of+Cell+Biology%22%5BJournal%5D)">Cellular invasion by Staphylococcus aureus involves a fibronectin bridge between the bacterial fibronectin-binding MSCRAMMs and host cell beta1 integrins.</a> European Journal of Cell Biology. 79 (10), 672-679 (2000).</li>
<li>Patti, J. M., Hook, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microbial+adhesins+recognizing+extracellular+matrix+macromolecules%5BTitle%5D)+AND+%22Current+Opinion+in+Cell+Biology%22%5BJournal%5D)">Microbial adhesins recognizing extracellular matrix macromolecules.</a> Current Opinion in Cell Biology. 6 (5), 752-758 (1994).</li>
<li>Massey, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fibronectin-binding+protein+A+of+Staphylococcus+aureus+has+multiple%2C+substituting%2C+binding+regions+that+mediate+adherence+to+fibronectin+and+invasion+of+endothelial+cells%5BTitle%5D)+AND+%22Cellular+Microbiology%22%5BJournal%5D)">Fibronectin-binding protein A of Staphylococcus aureus has multiple, substituting, binding regions that mediate adherence to fibronectin and invasion of endothelial cells.</a> Cellular Microbiology. 3 (12), 839-851 (2001).</li>
<li>Jashari, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Belgian+and+European+experience+with+the+European+Homograft+Bank+%28EHB%29+cryopreserved+allograft+valves--assessment+of+a+20+year+activity%5BTitle%5D)+AND+%22Acta+Chirurgica+Belgica%22%5BJournal%5D)">Belgian and European experience with the European Homograft Bank (EHB) cryopreserved allograft valves--assessment of a 20 year activity.</a> Acta Chirurgica Belgica. 110 (3), 280-290 (2010).</li>
<li>Cheatham, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clinical+and+hemodynamic+outcomes+up+to+7+years+after+transcatheter+pulmonary+valve+replacement+in+the+US+melody+valve+investigational+device+exemption+trial%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Clinical and hemodynamic outcomes up to 7 years after transcatheter pulmonary valve replacement in the US melody valve investigational device exemption trial.</a> Circulation. 131 (22), 1960-1970 (2015).</li>
<li>Que, Y. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fibrinogen+and+fibronectin+binding+cooperate+for+valve+infection+and+invasion+in+Staphylococcus+aureus+experimental+endocarditis%5BTitle%5D)+AND+%22The+Journal+of+Experimental+Medicine%22%5BJournal%5D)">Fibrinogen and fibronectin binding cooperate for valve infection and invasion in Staphylococcus aureus experimental endocarditis.</a> The Journal of Experimental Medicine. 201 (10), 1627-1635 (2005).</li>
<li>Veloso, T. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bacterial+adherence+to+graft+tissues+in+static+and+flow+conditions%5BTitle%5D)+AND+%22The+Journal+of+Thoracic+and+Cardiovascular+Surgery%22%5BJournal%5D)">Bacterial adherence to graft tissues in static and flow conditions.</a> The Journal of Thoracic and Cardiovascular Surgery. 155 (1), 325-332 (2018).</li>
<li>Liesenborghs, L., Verhamme, P., Vanassche, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Staphylococcus+aureus%2C+master+manipulator+of+the+human+hemostatic+system%5BTitle%5D)+AND+%22Journal+of+Thrombosis+and+Haemostasis%22%5BJournal%5D)">Staphylococcus aureus, master manipulator of the human hemostatic system.</a> Journal of Thrombosis and Haemostasis. 16 (3), 441-454 (2018).</li>
<li>Chiu, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Shear+stress+increases+ICAM-1+and+decreases+VCAM-1+and+E-selectin+expressions+induced+by+tumor+necrosis+factor-%5Balpha%5D+in+endothelial+cells%5BTitle%5D)+AND+%22Artheriosclerosis%2C+Thrombosis%2C+and+Vascular+Biology%22%5BJournal%5D)">Shear stress increases ICAM-1 and decreases VCAM-1 and E-selectin expressions induced by tumor necrosis factor-[alpha] in endothelial cells.</a> Artheriosclerosis, Thrombosis, and Vascular Biology. 24 (1), 73-79 (2004).</li>
<li>Jockenhoevel, S., Zund, G., Hoerstrup, S. P., Schnell, A., Turina, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cardiovascular+tissue+engineering%3A+a+new+laminar+flow+chamber+for+in+vitro+improvement+of+mechanical+tissue+properties%5BTitle%5D)+AND+%22ASAIO+Journal%22%5BJournal%5D)">Cardiovascular tissue engineering: a new laminar flow chamber for in vitro improvement of mechanical tissue properties.</a> ASAIO Journal. 48 (1), 8-11 (2002).</li>
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</ol>

Recent clinical observations give special awareness to IE as a complication in patients having undergone valve replacement of the RVOT6,13. Dysfunction of the implanted valve in IE is the result of bacterial interaction with the endovascular graft leading to extensive inflammatory and procoagulant reactions1,14. The presented novel in vitro model allowed us to investigate if differences in tissue...

Staphylococcus aureus (S. aureus) employs a variety of virulence strategies to circumvent the host immune defense system colonizing biological or non-biological surfaces implanted in the human circulation, which leads to severe intravascular infections such as sepsis and IE1,2,3,4,5. IE remains an important treatment associated complication in patients after implantation of prosthetic heart valves while individual factors contributing to the onset of IEare not yet fully understood6,7. Under flow conditions, bacteria encounter shear forces, which they need to overcome in order to adhere to the vessel wall8. Models, which allow studying the interplay between bacteria and prosthetic valve tissue or endothelium under flow, are of interest as they reflect the in vivo situation more.
Several specific mechanisms facilitate bacterial adherence to endothelial cells (ECs) and to the exposed subendothelial matrix (ECM) leading to tissue colonization and maturation of vegetations, being essential early steps in IE9. Various staphylococcal surface proteins or MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) have been described as mediators of adhesion to host cells and to ECM proteins by interacting with molecules such as fibronectin, fibrinogen, collagen and von Willebrand factor (VWF)8,10,11. However, in view of intra-molecular folding of some virulence factors, mostly studied in static conditions, many of these interactions may have different relevance in endovascular infections in circulating blood.
Therefore, we present an in-house designed in vitro parallel-plate flow chamber model, which allows the assessment of bacterial adherence to different components of ECM and ECs in the context of tissue grafts implanted in the RVOT position. The overall purpose of the method described in this work is to study mechanisms of interaction between bacteria and underlying endovascular tissues in flow conditions, which are closely related to the in vivo environment of bloodstream pathogens such as S. aureus. This novel approach focuses on the susceptibility of graft tissue surfaces to bacterial adherence to identify potential risk factors for the development of IE.

<table><tbody><tr><td>Bovine Pericardium (BP) patch, Supple Peri-Guard Pericardium</td><td>Synovis Surgical Innovations, USA</td><td>PC-0404SN</td><td></td></tr><tr><td>Bovine Jugular Vein conduits (BJV)</td><td>Contegra conduit; Medtronic Inc, USA</td><td>M333105D001</td><td></td></tr><tr><td>CH cryopreserved homograft</td><td>European Homograft Bank (EHB)</td><td>-</td><td></td></tr><tr><td>Acu-Punch</td><td>Acuderm Inc, USA</td><td>P850 (8 mm); P1050 (10 mm)</td><td></td></tr><tr><td>human Albumin</td><td>Flexbumin; Baxter, Belgium</td><td>BE171464&lt;br/&gt; LOT:16G12C</td><td></td></tr><tr><td>Tryptic soy broth (TSB)</td><td>Fluka, Steinheim, Germany</td><td>22092-500G</td><td></td></tr><tr><td>Heart infusion broth (BHI)</td><td>Fluka</td><td>53286-500G</td><td></td></tr><tr><td>Phosphate buffered saline (PBS).</td><td>Gibco</td><td>14190-094</td><td></td></tr><tr><td>5(6)-Carboxyfluorescein N-hydroxysuccinimide ester (CF)</td><td>Sigma-Aldrich, Germany</td><td>21878-100MG-F</td><td></td></tr><tr><td>Peristaltic pump (MODEL ISM444B)</td><td>Ismatec BVP-Z Standard; Cole Parmer, Wertheim, Germany</td><td>631942-2</td><td></td></tr><tr><td>Sonication bath</td><td>VWR Ultrasonic Cleaner; VWR, Radnor, Pa</td><td>142-6044</td><td>230V/50 -60Hz 60VA; HF45kHz, 30W</td></tr><tr><td>ProLong Gold Antifade Mountant</td><td>Invitrogen by ThermoFisher</td><td>P36930</td><td></td></tr><tr><td>InCell Analyzer 2000 (fluorescence scanner)</td><td>GE Healthcare Life Sciences, Pittsburgh, Pa</td><td>29027886</td><td></td></tr><tr><td>Arium Pro VF - ultrapure water - H&lt;sub&gt;2&lt;/sub&gt;O MilliQ</td><td>Millipore</td><td>87206462</td><td></td></tr><tr><td>Microscopic slides - Tissue Culture Chambers (1-well)</td><td>Sarstedt</td><td>94.6140.102</td><td></td></tr><tr><td>1-well on Lumox detachable</td><td>Sarstedt</td><td>94.6150.101</td><td></td></tr><tr><td>Stainless Steel - surgical Blades</td><td>Swann-Morton</td><td>311</td><td></td></tr><tr><td>Tygon Silicone Tubing, 1/8&quot;ID x 1/4&quot;OD</td><td>Cole-Parmer</td><td>EW-95702-06</td><td>Temperature range: &amp;ndash;80 to 200&amp;deg;C&lt;br/&gt; Sterilize: With ethylene oxide, gamma irradiation, or autoclave for 30 min, 15 psi of pressure</td></tr><tr><td>PharMed BPT Tubing</td><td>Saint-Gobain</td><td>AY242012</td><td>Autoclavable 30 min at 121&amp;deg;C</td></tr><tr><td>Tygon LMT-55 Tubing</td><td>Saint Gobain Performance Plastics&amp;trade;</td><td>15312022</td><td></td></tr><tr><td>Thermostat</td><td>BMG BIOMEDIZINTECHNIK</td><td>300-0042</td><td>230V, 90VA, 50Hz</td></tr></tbody></table>

an in vitro model of a parallel-plate perfusion system to study bacterial adherence to graft tissues

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart disease. When using prosthetic materials however, these grafts are susceptible to bacterial infections and various host responses.
Identification of bacterial and host factors that play a vital role in endovascular adherence of microorganisms is of importance to better understand the pathophysiology of the onset of infections such as infective endocarditis (IE) and to develop preventive strategies. Therefore, the development of competent models to investigate bacterial adhesion under physiological shear conditions is necessary. Here, we describe the use of a newly designed in vitro perfusion chamber based on parallel plates that allows the study of bacterial adherence to different components of graft tissues such as exposed extracellular matrix, endothelial cells and inert areas. This method combined with colony-forming unit (CFU) counting is adequate to evaluate the propensity of graft materials towards bacterial adhesion under flow. Further on, the flow chamber system might be used to investigate the role of blood components in bacterial adhesion under shear conditions. We demonstrated that the source of tissue, their surface morphology and bacterial species specificity are not the major determining factors in bacterial adherence to graft tissues by using our in-house designed in vitro perfusion model.

To better understand the mechanisms behind IE development, this model enables the evaluation of bacterial and tissue associated factors present in the in vivo situation of infection onset.
In detail, the novel in vitro approach allows to quantify bacterial adhesion in flow conditions to different graft tissues by perfusing fluorescently labeled bacteria over the tissues exerting the shear stresses in t...

We describe an in-house designed in vitro flow chamber model, which allows the investigation of bacterial adherence to graft tissues.

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart ...

an-vitro-model-parallel-plate-perfusion-system-to-study-bacterial

Research

JoVE Journal

Developmental Biology

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (&lt;30 μm) is described.  This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Source: Ditkowski, B., et al. An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues. J. Vis. Exp. (2019).This video demonstrates the method of using a parallel-plate flow chamber to perfuse fluorescently labeled bacteria over vascular graft tissues, enabling controlled analysis of bacterial adhesion and early pathogenesis under shear flow conditions.

In Vitro Parallel-Plate Flow Model to Study Bacterial Adhesion on a Tissue Biopsy

Source: Calatayud Arroyo, M., et al. Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface. J. Vis. Exp. (2018).This video demonstrates an in vitro gut host–microbe model to study bacterial viability. A co-culture of Caco-2 and HT29-MTX cells is incubated with bacteria pre-treated under simulated digestive conditions. Epithelial barrier integrity is assessed using transepithelial electrical resistance, and fractions from the medium, solubilized...

In Vitro Gut Model to Assess Bacterial Adhesion and Internalization

Source: Claes, J., et al, In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions. J. Vis. Exp. (2015).This video demonstrates a method for studying host-pathogen interactions using a micro-parallel plate flow chamber seeded with human primary endothelial cells. Following endothelial cell activation by an ionophore and perfusion with fluorescently labeled bacteria, the cells release von Willebrand factor (VWF), which binds bacterial adhesins under shear...

Micro-Parallel Plate Flow Chamber Model for Studying Bacterial Adhesion to Human Endothelial Cells

Source: Claes, J., et al, In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions. J. Vis. Exp. (2015).This video demonstrates an in vivo method for analyzing shear-dependent bacterial adhesion in a live mouse model. Following surgical preparation and catheterization, the mesenteric vasculature is exposed for real-time imaging under a fluorescence microscope. Perfusion with an ionophore and fluorescently labeled bacteria reveals how shear stress triggers...

An In Vivo Model to Study Shear-Dependent Bacterial Adhesion

Neisseria meningitidis is a human specific pathogen that infects blood vessels. In this protocol human microvessels are introduced into a mouse by grafting human skin onto immunocompromised mice. Bacteria adhere extensively to the human vessels, leading to vascular damage and development of the purpuric rash typically observed in human cases.

Humanized Mouse Model to Study Bacterial Infections Targeting the Microvasculature

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

Leukocytes avidly interact with vascular cells and platelets after vessel wall injury or during inflammation. Here, we describe a straightforward laminar flow-based assay to characterize the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners.

Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets

This protocol demonstrates methods to enable extended in vitro culture of patient-derived xenografts (PDX). One step enhances overall viability of multicellular cluster cultures in 3D hydrogels, through straightforward removal of non-viable single cells. A secondary step demonstrates best practices for PDX culture in a perfused microfluidic platform.

Enhanced Viability for Ex vivo 3D Hydrogel Cultures of Patient-Derived Xenografts in a Perfused Microfluidic Platform

This protocol presents the setup and operation of a newly developed, 3D printed bioreactor for the ex vivo culture of blood vessels in perfusion. The system is designed to be easily adopted by other users, practical, affordable, and adaptable to different experimental applications, such as basic biology and pharmacological studies.

Ex Vivo Perfusion Culture of Large Blood Vessels in a 3D Printed Bioreactor

Learn an in vitro flow chamber model to study bacterial adherence to graft tissues for infective endocarditis research. This method reveals molecular interactions driving tissue infectiveness under standardized conditions.

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

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