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

Summary

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

Abstract

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.

Introduction

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 IE^1,2,3,4,5. 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 understood^6,7. 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.

Protocol

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.

1. Rinse all tissues with 0.9% NaCl prior to use.
2. Prepare tissue biopsies using a disposable skin biopsy punch to cut circular tissue pieces (10 mm in diameter).
3. Cut all tissue patches to the same height using disposable sterile scalpels.
4. For tissues fixed with glutaraldehyde (for example BJV conduit), incubate graft pieces overnight at 4 °C with 200 g/L of human albumin to neutralize the fixative.
5. Wash out residues of glutaraldehyde with 0.9% NaCl in a microtiter plate.  Repeat 3 times for 1 minute.

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 °C in tryptic soy broth (TSB) and S. sanguinis was grown at 37 °C with 5% CO₂ in brain heart infusion broth (BHI).

1. Prepare overnight culture of bacteria on a solid blood agar plate.
   1. Use a sterile loop to scrape the frozen bacteria off and inoculate onto a Mueller-Hinton blood agar plate for overnight culture at 37 °C.
2. 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 mL tube and culture overnight at 37 °C.
3. Centrifuge overnight cultures (3000 x g, 4 °C, 10 min) and resuspend the pellets in 10 mL of phosphate buffered saline (PBS). Place 14 mL tubes on ice.
4. 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 °C. Further dilute the stock of CF to 150 µg/mL using 'ultrapure' water.
   Note: Protect tubes from light using aluminum foil and store at -20 °C.
5. Centrifuge the bacteria (3000 x g, 4 °C, 10 min) and resuspend the pellets in 800 µL of PBS and add 200 µL of the 150 µg/mL CF solution (final concentration of 30 µg/mL used for perfusion experiments). Protect tubes from light with aluminum foil and incubate for 30 min using an orbital shaker.
6. After labeling, block with 2% of bovine serum albumin (BSA) solution in PBS and spin (3000 x g, 4 °C, 10 min). Follow with a wash step using 10 mL of PBS and pellet bacteria by centrifugation (3000 x g, 4 °C, 10 min).
7. 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.

3. In vitro Perfusion Experiments using a Parallel-Plate Flow Chamber

1. 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.
   1. 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.
   2. Insert the holder with the tissue into the gasket sheet that is embedded in the bottom metal frame of the chamber. 
   3. 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.
2. Connect the flow chamber with a peristaltic pump and the fluid reservoir with the tubes.
3. 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 °C using a plate thermostat (Table of Materials).
4. Recirculate continuously the 100 mL bacterial suspension using the same collection reservoir.
5. After perfusion, dismantle the chamber to release the graft and wash the tissue piece two times with 4 mL of PBS 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.
6. Place each tissue biopsy into a separate 14 mL tube containing 1 mL of sterile 0.9% NaCl. Label the tube as #1.
7. 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 °C in TSB liquid medium followed by OD₆₀₀ measurements compared to control patches treated with a bacteria free solution.
8. Use a serial dilution method on Mueller-Hinton blood agar plates to count CFUs.
   1. Prepare a single 14 mL tube with 10 mL of sterile saline to make 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.
   2. Prepare three 14 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.
   3. Vortex mix each tube for 15 s. Vortex the tubes with the tissue biopsy as well as the initial bacterial suspension to make serial dilutions.
   4. 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.
   5. 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 bacterial perfusates, label the 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.
   6. To continue preparing the serial dilutions, transfer 100 µL of tube #1 to tube #2 and mix vigorously with vortex.
   7. Spread 100 µL of the contents of tube #1 and #2 onto the corresponding sectors 10^-1 and 10^-3 of the agar plate. Likewise, spread 10 µL of tube #2 on the sector 10^-4, repeat this step 4 times to obtain 4 separate growths from each volume of 10 µL.
      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.
   8. To prepare the serial dilutions of the initial culture, transfer 100 µL of bacterial suspension from step 2.7 to tube #3 and mix vigorously with vortex. Add 100 µL of tube #3 to tube #4 and mix well, repeat the procedure for subsequent tube #5.
   9. Spread 100 µL 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.
   10. 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 °C for overnight incubation.
   11. After overnight incubation, count the bacterial colonies to obtain the number of CFUs resulting from the adhesion to the tissue biopsies as well as CFUs/mL in the starting bacterial suspension used for the perfusion. Express results as CFU/cm².

4. Fluorescence Microscopy of Adhered Bacteria to Graft Tissues upon Perfusion

1. 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.
2. Prepare a 6-well plate and place droplets of mounting medium (Table of Materials).
3. Place each piece of tissue with its perfused surface downward on a single drop of mounting medium.
4. 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.

Results

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...

Discussion

Recent clinical observations give special awareness to IE as a complication in patients having undergone valve replacement of the RVOT^6,13. Dysfunction of the implanted valve in IE is the result of bacterial interaction with the endovascular graft leading to extensive inflammatory and procoagulant reactions^1,14. The presented novel in vitro model allowed us to investigate if differences in tissue...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bovine Pericardium (BP) patch, Supple Peri-Guard Pericardium	Synovis Surgical Innovations, USA	PC-0404SN
Bovine Jugular Vein conduits (BJV)	Contegra conduit; Medtronic Inc, USA	M333105D001
CH cryopreserved homograft	European Homograft Bank (EHB)	-
Acu-Punch	Acuderm Inc, USA	P850 (8 mm); P1050 (10 mm)
human Albumin	Flexbumin; Baxter, Belgium	BE171464 LOT:16G12C
Tryptic soy broth (TSB)	Fluka, Steinheim, Germany	22092-500G
Heart infusion broth (BHI)	Fluka	53286-500G
Phosphate buffered saline (PBS).	Gibco	14190-094
5(6)-Carboxyfluorescein N-hydroxysuccinimide ester (CF)	Sigma-Aldrich, Germany	21878-100MG-F
Peristaltic pump (MODEL ISM444B)	Ismatec BVP-Z Standard; Cole Parmer, Wertheim, Germany	631942-2
Sonication bath	VWR Ultrasonic Cleaner; VWR, Radnor, Pa	142-6044	230V/50 -60Hz 60VA; HF45kHz, 30W
ProLong Gold Antifade Mountant	Invitrogen by ThermoFisher	P36930
InCell Analyzer 2000 (fluorescence scanner)	GE Healthcare Life Sciences, Pittsburgh, Pa	29027886
Arium Pro VF - ultrapure water - H2O MilliQ	Millipore	87206462
Microscopic slides - Tissue Culture Chambers (1-well)	Sarstedt	94.6140.102
1-well on Lumox detachable	Sarstedt	94.6150.101
Stainless Steel - surgical Blades	Swann-Morton	311
Tygon Silicone Tubing, 1/8"ID x 1/4"OD	Cole-Parmer	EW-95702-06	Temperature range: –80 to 200°C Sterilize: With ethylene oxide, gamma irradiation, or autoclave for 30 min, 15 psi of pressure
PharMed BPT Tubing	Saint-Gobain	AY242012	Autoclavable 30 min at 121°C
Tygon LMT-55 Tubing	Saint Gobain Performance Plastics™	15312022
Thermostat	BMG BIOMEDIZINTECHNIK	300-0042	230V, 90VA, 50Hz