Name: an in vitro model of a parallel-plate perfusion system to study bacterial adherence to graft tissues
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues at JoVE.com

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 instructions<sup cla...

<ol>
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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 
			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 - H2O 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&#34;ID x 1/4&#34;OD</td>
			<td>Cole-Parmer</td>
			<td>EW-95702-06</td>
			<td>Temperature range: &#8211;80 to 200&#176;C 
			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&#176;C</td>
		</tr>
		<tr>
			<td>Tygon LMT-55 Tubing</td>
			<td>Saint Gobain Performance Plastics&#8482;</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

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...

Watch this Scientific Journal Video about An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues at JoVE.com

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

We describe an in-house designed in vitro flow chamber model, which allows the investigation of 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 ...

This method can help answer key questions in the pathogenesis of infective endocarditis, and pathobiograms through fields such as the interplay between bacteria, blood cells, and endothelial cells lining musculature. The main advantage of this technique is that the tissue grafts might be directly exposed to mutual interactions with various targets, such as bacteria, platelets, and proteins in standardized flow conditions. The implication of this technique extend to our therapy of infective endocarditis, because it can unravel important molecular interactions and factors driving high infectiveness of certain tissues.This method provides insight into pathogenesis of vegetation formation. It can also be applicable in studies investigating vascular permeability, cell mobility, and gene cell expression. To begin the protocol, place a previously prepared tissue biopsy 10 millimeters in diameter, and the same thickness between a microscope slide and an eight millimeter circular perforation, and a rubber gasket with the inner surface facing up, to contact the bacterial suspension.Focus on the tissue graft orientation while placed in the holder. Make sure the inner surface of the tissue is already exposed to the bloodstream faces up to contact your experimental medium. Next, insert your holder with the tissue into the gasket sheet that is embedded in the bottom metal frame of the chamber.Attach the upper metal frame with the corresponding gasket sheet onto the bottom of the chamber with the previously inserted tissue holder. Then mount the entire chamber with eight screws and screw nuts. Make sure that the chamber height is always the same across grafts by using a caliper or ruler.Next connect the flow chamber with a peristaltic pump. Then connect the 400 milliliter fluid reservoir with the tubes. Perfuse the tissues with 100 milliliter suspensions of ten to the seven fluorescently-labeled colony forming units in phosphate buffered saline with the sheer stress of three dines per square centimeter and corresponding flow rate of four milliliters per minute for one hour using the peristaltic pump and the bacterial reservoir.Recirculate continuously the 100 milliliter bacterial suspension using the same collection reservoir. After perfusion, dismantle the chamber to release the graft. Wash the tissue piece two times with four milliliters of PBS in a 12-well plate using an orbital shaker for three minutes.Then cut the inner part of the graft using a skin biopsy punch with a smaller diameter. Place each tissue biopsy into a separate 14 milliliter tube containing one milliliter of sterile 0.9%sodium chloride. Detach the bacteria from the tissue using a sonication bath for 10 minutes.Prepare a single 14 milliliter tube with 10 milliliters of sterile 0.9%sodium chloride to make serial dilutions of the bacterial suspension obtained after sonication. Label the tube number two. Prepare three 14 milliliter tubes with 10 milliliters of sterile 0.9%sodium chloride for serial dilutions of the initial bacterial suspension taken to the profusion experiment.Label the tubes number three, number four, and number five. Next, secure three agar plates, one for the bacterial perfusion, one for the control perfusion of PBS, and the third one for the initial bacterial suspension used for perfusions. 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 colony-forming units in the bacterial perfusing, label the plate as follows:10-1, 10-3, 10-5, and 10-7. Vortex the tubes containing the tissue biopsy for 15 seconds each, to make serial dilutions. To prepare the serial dilutions, transfer 100 microliters of tube number one to tube number two, and vortex the tube to thoroughly mix the solution.Spread 100 microliters of the contents of tube number one and number two onto the corresponding sectors, 10-1 and 10-3 of the agar plate. Then spread 10 microliters of tube number two on sector 10 four. Repeat this step four times to obtain four separate growths from each volume of 10 microliters.To prepare serial dilutions of the initial culture, first vortex the dilution for 15 seconds and transfer 100 microliters of bacterial suspension to tube number three, and mix vigorously by vortexing. Add 100 microliters of tube number three to tube number four and mix again. Repeat the procedure for tube number five from tube number four.Spread 100 microliters of the contents of tubes number three, number four, number five, and the labeled initial culture respectively onto sectors 10-3, 10-5, 10-7, and 10-1 of the blood agar plate. Leave the blood agar plates under the laminar hood to air dry the bacterial spreads, typically for 10 minutes. Afterwards, place the plates at 37 degrees Celsius for overnight incubation.After overnight incubation, count the bacterial colonies to obtain the number of CFUs. Under sheer stress conditions, similar bacterial attachment across the various graft tissues was observed for both staphylococcus aureus and staphylococcus epidermidis infection. Following the procedures, streptococcus sanguinis displayed significant reduction of adherence to the bovine jugular vein wall, when compared to the bovine pericardium patch.When comparing the three species of bacteria, streptococcus sanguinis presents significantly lower adhesion to the bovine jugular vein wall in relation to staphylococcus aureus and staphylococcus epidermidis. While attempting this procedure, it's important to remember to have prepared correct tissues of the same head. It will ensure similar conditions across all experiment specimens and minimize data variability between different series of experiments.After its development, this technique allows researchers to explore the complex disorder systems in a step by step manner by enriching their in vitro model approaches with different elements to build up full complexity close to medical status.

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