This work was funded by the National Institutes of Health, Grant R01HL13334. The authors would like to thank Dr. Kevin Haworth for assisting with Drabkin&#39;s assay and Dr. Viktor Bollen for his support in designing the protocol. The authors are also thankful to Dr. Adam Maxwell for his guidance on designing the histotripsy source.

For the results presented here, venous human blood was drawn to form clots after approval from the local internal review board (IRB #19-1300) and written informed consent provided by volunteer donors24. This section outlines&#160;a design protocol to assess lysotripsy efficacy. The protocol is based on a previous work by Bollen et al.24.
1. Clot modeling
NOTE: Prepare the clots within 2 weeks but more than 3 days prior to the day of the experiment to ensure clot stability and maximize retraction28. Prepare the clot following the approval from local institutional review board.
<ol>
	<li>Prepare borosilicate Pasteur pipette for storing the blood (see Table of Materials for specifications of the pipette). Borosilicate tubes are used because of the hydrophilic nature of the material which promotes platelet activation and clot retraction29. Seal the tip of the pipette via heating over a Bunsen burner.</li>
	<li>Draw fresh human venous blood. Aliquot the total blood drawn in 2 mL increments per desired clot. Transfer each 2 mL aliquot to one Pasteur pipette. 
	NOTE: Execute step 1.2 within approximately 3 min of blood draw so that blood does not clot before transferring to pipettes.&#160;Also, ensure the volunteer donor is not on any medications that may alter the clotting cascade (e.g. blood thinners or platelet inhibitors).</li>
	<li>Incubate the blood aliquots within the pipettes (equal to the number of clots required) in a water bath for 3 h at 37 &#176;C.</li>
	<li>Store the pipettes for a minimum of 3 days at 4 &#176;C to allow for retraction of clots28. As the clots retract, serum will be observed to accumulate at the top of the clot within the pipette. The rt-PA response of clots remains stable for 2 weeks following retraction28.</li>
</ol>
2. Water tank preparation
<ol>
	<li>Fill the water tank with reverse osmosis water. Line the bottom surface of the tank with an acoustic absorbing material to reduce reflection of the therapy or imaging ultrasound pulses. Use a water handling system to degas and filter the water to minimize bubble nuclei. 
	NOTE: One way to filter the water is using an inline filter. The specifications of the bag used for generating the representative results is given in the Table of Materials.</li>
	<li>Place two heating elements at the bottom surface of the tank. Heat the water to 37.3 &#730;C to achieve the maximum lytic enzymatic activity30.</li>
	<li>Setup a flow channel as shown in Figure 1A. The flow channel consists of tubing, a model vessel with material and geometric properties representative of an iliofemoral vein, a reservoir for plasma, and a syringe on the distal most end of the reservoir (Table of Materials). The syringe is connected to a pump to regulate a flow through the channel during the experiment.</li>
	<li>Manually submerge the flow channel in the water tank to bring the channel to physiologic temperature during the degassing/filtering/heating stage (steps 2.1 and 2.2).</li>
</ol>
3. Preparation of plasma and rt-PA mixture
<ol>
	<li>Prior to experiment day 
	&#8203;NOTE: When stored at -80 &#176;C, plasma is stable for at least 2.5 years31 and rt-PA is stable for at least 7 years32. Therefore, execute step 3.1 anytime within these periods to ensure stability of the two components.
	<ol>
		<li>Dilute the rt-PA obtained from a manufacturer in powdered form to 1 mg/mL in sterile water.</li>
		<li>Aliquot 100 &#181;L of diluted rt-PA into 0.5 mL centrifuge tubes and store them at -80 &#176;C.</li>
		<li>Aliquot 35 mL of human fresh-frozen type O plasma in 50 mL centrifuge tubes. Store the tubes at -80 &#176;C.</li>
	</ol>
	</li>
	<li>On the experiment day
	<ol>
		<li>Retrieve plasma aliquots from the freezer. Retrieve as many aliquots as the number of clots to be tested that day. Immerse the frozen aliquots in a water bath at 37 &#176;C to thaw (~10 min).</li>
		<li>Once plasma has thawed, pour it into a beaker that is triply rinsed with ultrapure water. Lightly cover the mouth of the beaker with aluminum foil to prevent contamination and place the beaker in the water bath. Allow the foil to be loose enough to allow air to contact the plasma.</li>
		<li>Let plasma equilibrate at 37 &#176;C to atmospheric pressure for at least 2 h.</li>
		<li>Take out the frozen rt-PA vials and place on ice until needed, with one vial for each experiment run.</li>
		<li>Make low gelling agarose (2%) in a 50 mL flask, by dissolving agarose in ultrapure water. Choose the total amount of agarose solution such that approximately 2 mL is available for each specimen to be analyzed. Heat the solution in flask in a microwave until bubbly. Secure the flask with a waterproof screw lid on it. Submerge the flask in the water bath alongside the plasma. 
		&#8203;NOTE: This step ensures agarose is available to secure exposed clot segments for histology analysis following histotripsy insonation.</li>
	</ol>
	</li>
</ol>
4. Setting up histotripsy source and imaging array
<ol>
	<li>Ensure the motorized positioners can be controlled from the runtime environment of a programming platform, using directions and commands provided by the manufacturer. Check that the motors of the system are connected to the appropriate port of the computer with the runtime environment.</li>
	<li>Mount the histotripsy source on the motorized positioning system as shown in Figure 1B.</li>
	<li>Connect the histotripsy source to its driving electronics (e.g., power amplifier and function generator) via the appropriate connectors (e.g., BNC cables) as specified by the manufacturer. 
	NOTE: Ensure that the driving electronics of the histotripsy source can be controlled via the runtime environment used in step 4.1.</li>
	<li>Cover the imaging array with a probe cover and affix the array coaxially in the aperture of the histotripsy source as shown in Figure 2. Be sure to understand the orientation of the imaging plane relative to the orientation of the histotripsy source.</li>
	<li>Connect the imaging array to an ultrasound scanning system. Ensure that this system can control the operation and triggering of the imaging array, and collect imaging data, as per the commands provided by the manufacturer of the scanner.</li>
	<li>Submerge the histotripsy source/imaging array into the tank while degassing as shown in Figure 1A. Gently remove any air bubbles using a syringe from the surface of the histotripsy source or imaging array. 
	NOTE: Immerse the histotripsy source and imaging array completely in water before operation. Avoid touching the surface of the histotripsy source.</li>
	<li>Acquire B-mode images at a rate of 20 frames per second using the imaging array and the built-in commands of the scanner. Adjust the imaging window to ensure visualization of the focus of the histotripsy source in these real-time images. 
	NOTE: It is assumed that the dimensions of the focal region of the therapeutic source are known.</li>
	<li>Set the operating parameters of the histotripsy source, including fundamental frequency (e.g., 1.5 MHz), pulse repetition frequency (e.g., 20-100 Hz), pulse duration (e.g., 1-20 cycles per pulse), and total number of pulses per location (e.g., 100-2,000)18,23,24,33. Modify these parameters if sufficient clot lysis is not achieved or if the bubble activity extends beyond the lumen of the model vessel. To set these parameters, use the protocol provided by the manufacturer of the source or use a programming platform that can communicate with the source (Step 4.3).</li>
	<li>Using the manufacturer&#39;s protocol or programming platform used in step 4.8, run the histotripsy source at the set parameters in degassed water only, without any obstruction in the surrounding environment. Increase the voltage applied to the histotripsy source until a bubble cloud is formed.</li>
	<li>Using the real-time imaging of step 4.7, adjust the position of the imaging array inside the confocal transducer opening until the bubble cloud is located approximately at the center of the image window. The bubble cloud is the region of hyperechoic pixels in the imaging plane (Figure 3). Tighten the screws to hold the imaging array firmly in the transducer opening. 
	NOTE: If the array is aligned properly, the azimuthal position of the bubble cloud should be approximately at 0 mm in the imaging plane. The imaging array may project slightly from the inner surface of the therapy source, and therefore the range position of the bubble cloud may differ from the focal length of the source.</li>
	<li>Identify the bubble cloud location in the imaging plane. Assign the focus of the histotripsy source as the center of the bubble cloud (Figure 3).</li>
	<li>Record the detected focal location (step 4.11) in the imaging window (Figure 3). A possible way to mark the focal position is placing a cursor to note the location in the imaging window, if available with the imaging platform.</li>
	<li>Discontinue insonation and set the voltage applied to the histotripsy source to 0 V.</li>
</ol>
5. Clot preparation
<ol>
	<li>Remove the clot from the pipette by cutting the sealed end with pliers. Let the clot slide into a Petri dish along with the serum. If the clot does not dislodge, gently apply pressure from the other end of the pipette via saline flush to remove the clot.</li>
	<li>Cut the clot to 1 cm length using a scalpel, aiming for a uniform piece from the center (i.e., away from sections of the clot formed at the top or bottom of the pipette).</li>
	<li>Use a cleaning wipe to blot the cut section of the clot gently to remove excess fluid.</li>
	<li>Using tweezers, place the clot section gently on a weighing scale and record the weight.</li>
	<li>Manually raise the flow channel out of the water tank and remove the model vessel from the flow channel.</li>
	<li>Place the clot in the model vessel using tweezers and attach the model vessel again to the flow channel. 
	NOTE: A nylon rod can be placed within the model vessel to prevent the clot from moving downstream due to the flow.</li>
	<li>Lower the flow channel into the tank in such a way that the proximal end of the stage relative to the reservoir is low compared to the distal side. The angling of the stage in this manner prevents trapping of bubbles in the model vessel when plasma is drawn through the flow channel in step 6.1.</li>
	<li>Add 30 mL of plasma to the reservoir using a pipette and monitor the temperature until it reaches at least 36 &#176;C.</li>
	<li>Use a pipette to dispense the rt-PA (80.4 &#181;g in 30 mL of plasma, 2.68 &#181;g/mL) into the plasma reservoir. Stir the plasma with the pipette to ensure a uniform rt-PA distribution within the reservoir.</li>
</ol>
6. Priming the flow channel
<ol>
	<li>Draw plasma into the flow channel from the reservoir via the syringe pump until the plasma fills the model vessel. 
	NOTE: If the clot is not flush with the nylon rod, use short pump draws at 60 mL/min to try to force the plasma downstream the clot or manually draw through the syringe. Limit the amount of plasma drawn in this process or refill the reservoir using additional plasma/rt-PA to ensure 30 mL of solution in the flow channel.</li>
	<li>Using the motorized positioners, align the imaging array parallel to the length of the clot using the imaging script (step 4.7). The parallel alignment enables the user to visually ensure proper placement of the clot and absence of bubbles inside the model vessel.</li>
	<li>Level the model vessel manually and visually ensure that no air bubbles are present using the imaging window (step 4.7).</li>
</ol>
7. Experiment procedure
<ol>
	<li>Pre-treatment 
	&#8203;NOTE: This step is to plan a path for the histotripsy source/imaging array for uniform histotripsy exposure along clot length.
	<ol>
		<li>Align the imaging array using the motorized positioners such that the imaging plane is parallel to the cross-section of the clot (i.e., perpendicular to the orientation described in step 6.2).</li>
		<li>Under guidance via the imaging window (step 4.7), move the histotripsy source to the proximal end of the clot relative to the reservoir using the motorized positioners. At this point, adjust the histotripsy source position such that the marked focal point in step 4.12 aligns with the center of the clot.</li>
		<li>Determine the insonation path along the clot length. To define this path, set three waypoints along the length of the clot (i.e., positions of the motors where histotripsy bubble activity is contained within the clot) in 5 mm increments. Align the waypoints such that the overall motion of the histotripsy source along the path is antegrade with flow in the system (i.e., the first waypoint is at the most proximal end of the clot relative to the reservoir, and the third waypoint is in the distal position relative to the reservoir).</li>
		<li>Prior to finalizing each waypoint, fire test pulses from the histotripsy source with the same insonation parameters as step 4.8 but reduce the overall exposure to 10 total pulses. Adjust the position of the histotripsy source using the motorized positioners if necessary, to contain bubble activity within the clot.</li>
		<li>At each waypoint, save the motor positions using the commands provided by the manufacturer, similar to the step 4.1.</li>
	</ol>
	</li>
	<li>Treatment 
	&#8203;NOTE: This step defines the procedure to treat the clot along its length according to the path defined in the pre-treatment step.
	<ol>
		<li>Run the syringe pump at 0.65 mL/min and wait for meniscus of the plasma to move. This flow rate mimics a near total occlusion of the iliofemoral vasculature24,34.</li>
		<li>Interpolate the path created in step 7.1.3 with intermediate steps between the established waypoints with a fixed step size (e.g., 0.5 mm). The step size is chosen to be smaller than half the width of the focal region as measured along the clot length (elevational direction of the imaging array). Move the histotripsy source using motorized positioners at each path location with insonation parameters defined in step 4.8.</li>
		<li>Monitor/image bubble activity during application of the histotripsy pulse at each path location using the imaging window (step 4.7). Center the image on the histotripsy focus with the image dimensions covering 15 mm in the azimuth and range. Prior to the application of histotripsy pulse at each location, acquire a B-mode image to provide visualization of the clot and model vessel, by creating a script in a programming platform. Ensure the script establishes communication with the scanner using the manufacturer&#39;s commands.</li>
		<li>During the application of the histotripsy pulse, implement the acquisition of acoustic emissions in the script in step 7.2.3 to form passive cavitation images post hoc35. Acquire one passive cavitation image after every 10 treatment pulses. Apply a flat time gain compensation of 50 at 8 incremental depths till the end of the imaging depth. Choose an appropriate acquisition window size such that the entire signal from the clot is captured with minimum loss of energy due to windowing35.</li>
		<li>If off-target bubble clouds are present, adjust the transducer position in situ with the motorized positioners. 
		&#8203;NOTE: Monitor for missed triggers of the imaging array. Adjust the number of acquired imaging data sets to ensure the storage of data is completed before subsequent triggering.</li>
	</ol>
	</li>
</ol>
8. Post experiment procedure
<ol>
	<li>Manually raise the model vessel out of the water tank to drain the perfusate via gravity. Be sure to keep the flow channel levelled to prevent the clot from moving downstream and out of the model vessel during draining.</li>
	<li>Collect the entire perfusate for further analysis in a small beaker (Figure 4A) by drawing plasma solution from the flow channel through the syringe pump at a very low flow rate.</li>
	<li>Disconnect the model vessel and remove the clot. If necessary, inject a small amount of saline into the model vessel to gently dislodge the clot.</li>
	<li>Wipe the clot similar to step 1.2.3. Weigh the clot on the weighing scale for assessing the clot mass loss.</li>
	<li>To analyze the D-dimer content, add 100 mg of aminocaproic acid to a microcentrifuge tube followed by 1 mL of perfusate, and mix well using a pipette. Perform a latex immune-turbidimetric assay to quantify the D-dimer within the sample36.</li>
	<li>To assess hemolysis, add 1 mL of perfusate to centrifuge tubes and spin at 610 x g (3,500 rpm) for 10 min. Combine 0.5 mL of supernatant (concentrate) with 0.5 mL of Drabkins solution and let the mixture rest at room temperature for 15 min. Transfer 200 &#181;L to well plates, as shown in Figure 4B. Use a plate reader to read absorbance at 540 nm, Figure 4C (Drabkins assay37).</li>
	<li>Histology assessment
	<ol>
		<li>Cut a section from the center of the clot of about 2-3 mm in length with a scalpel.</li>
		<li>Add the section to a cassette. Maintain orientation of the section relative to the direction of histotripsy pulse propagation.</li>
		<li>Add 2 mL of low gelling agarose solution prepared in step 3.2.5 into the cassette to fix the clot in place.</li>
		<li>Fix the sample in 10% formalin for 24 h. Place the sample in 70% alcohol after 24 h and perform standard hemotoxylin-eosin staining38.</li>
	</ol>
	</li>
</ol>
9. Passive cavitation image analysis
<ol>
	<li>Process the signals acquired from the imaging array during the histotripsy excitation (step 7.2.4) using an algorithm based on the robust Capon beamformer39 to create an image of acoustic emissions generated by the bubble cloud at each treatment location. 
	NOTE: To generate quantitative images, follow the steps described in Haworth et al.35. Otherwise, each pixel value in the image should represent the relative bubble cloud acoustic energy (units of V2) at each corresponding location.</li>
	<li>Segment the B-mode image aquired in steps 7.2.3 to distinguish between the pixels representing clot and the model vessel.</li>
	<li>Co-register the passive cavitation image with the B-mode image as shown in Figure 5B. Sum up the acoustic energy within the clot over the exposure period35.</li>
</ol>

The authors have nothing to disclose.

The proposed protocol presents a model to quantify treatment efficacy of lysotripsy. While the key details have been discussed, there are certain critical aspects to consider for the success of this protocol. The enzymatic activity of rt-PA has an Arrhenius temperature dependence30. Temperature is also a contributing factor to the speed of sound in water and tissue, and variations in temperature can cause minor alterations of the focal zone geometry. Thus, the water temperature should be carefully...

Thrombosis is the condition of clot formation in an otherwise healthy blood vessel that obstructs circulation1,2. Venous thromboembolism has an annual healthcare cost of $7-10 billion, with 375,000-425,000 cases in the United States3. Pulmonary embolism is the obstruction of the pulmonary artery and is the most serious consequence of venous thromboembolism. The primary source of pulmonary obstruction is deep vein thrombi, primarily from iliofemoral venous segments4,5,6. Deep vein thrombosis (DVT) has inherent sequela besides pulmonary obstructions, with long term complications that result in pain, swelling, leg ulcerations, and limb amputations7,8,9. For critical obstructions, catheter directed thrombolytics (CDT) are the frontline approach for vessel recanalization10. The outcome of CDT depends on a number of factors, including thrombus age, location, size, composition, etiology, and patient risk category11. Moreover, CDT is associated with vascular damage, infections, bleeding complications, and long treatment time10. Next generation devices aim to combine mechanical thrombectomy with thrombolytics (i.e., pharmacomechanical thrombectomy)12,13. Use of these devices lower the lytic dosage leading to reduced bleeding complications, and shortened treatment time12,13,14 as compared to CDT. These devices still retain issues of hemorrhagic side-effects and incomplete removal of chronic thrombi15. An adjuvant strategy is thus needed that can remove the thrombus completely with lower bleeding complications.
One potential approach is histotripsy-aided thrombolytic treatment, referred to as lysotripsy. Histotripsy is a non-invasive treatment modality that uses focused ultrasound to nucleate bubble clouds in tissues16. Bubble activity is generated not via exogenous nuclei, but by the application of ultrasound pulses with sufficient tension to activate nuclei intrinsic to tissues, including clot17,18. The mechanical oscillation of the bubble cloud imparts strain to the clot, disintegrating the structure into acellular debris19. Histotripsy bubble activity provides effective degradation of retracted and unretracted blood clots both in vivo and in vitro20,21,22. Prior studies have23,24 demonstrated that the combination of histotripsy and the lytic&#160;recombinant tissue-type plasminogen activator (rt-PA)&#160;significantly increases treatment efficacy compared to lytic alone or histotripsy alone. It is hypothesized that two primary mechanisms associated with histotripsy bubble activity are responsible for the improved treatment efficacy: 1) increased fibrinolysis due to enhanced lytic delivery, and 2) hemolysis of red blood cells within the clot. The bulk of the clot mass is comprised of red blood cells24, and, therefore, tracking erythrocyte degradation is a good surrogate for ablation of the sample. Other formed clot elements are also likely disintegrated under histotripsy bubble activity but are not considered in this protocol.
Here, a benchtop approach to treat DVT in vitro with lysotripsy is outlined. The protocol describes critical operating parameters of the histotripsy source, assessment of treatment efficacy, and image guidance. The protocol includes designing a flow channel to mimic an iliofemoral venous segment and manufacturing human whole blood clots. The experimental procedure outlines the positioning of the histotripsy source and imaging array to achieve histotripsy exposure along the clot placed in the flow channel. Relevant insonation parameters to attain clot disruption and minimize off-target bubble activity are defined. The use of ultrasound imaging for guidance and assessment of bubble activity is illustrated24. Metrics to quantify treatment efficacy such as clot mass loss, D-dimer (fibrinolysis), and hemoglobin (hemolysis) are outlined23,24,25,26,27. Overall, the study provides an effective means for executing and assessing the efficacy of lysotripsy to treat DVT.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Absorbing sheets</td>
			<td>Precision acoustics</td>
			<td>F28-SMALL-M</td>
			<td>300mm x 300 mm x 10 mm</td>
		</tr>
		<tr>
			<td>Borosilicate Pasteur pippettes</td>
			<td>Fisher Scientific</td>
			<td>1367820A</td>
			<td>14.6 cm length, 2 mL capacity</td>
		</tr>
		<tr>
			<td>Centrifuge tubes</td>
			<td>Eppendorf</td>
			<td>22364111</td>
			<td>1.5 mL capacity</td>
		</tr>
		<tr>
			<td>Drabkin&#39;s assay</td>
			<td>Sigma Aldrich</td>
			<td>D5941-6VL</td>
			<td></td>
		</tr>
		<tr>
			<td>Draw syringe</td>
			<td>Cole-Parmer</td>
			<td>EW-07945-43</td>
			<td>60 mL capacity</td>
		</tr>
		<tr>
			<td>Filter bags</td>
			<td>McMaster-Carr</td>
			<td>5162K111</td>
			<td>Remove particle size upto 1 microns</td>
		</tr>
		<tr>
			<td>Flow channel tubing</td>
			<td>McMaster-Carr</td>
			<td>5154K25</td>
			<td>Polyethylene-lined EVA plastic tubing (Outer diameter: 3/8&#34;, Inner diameter: 1/4&#34;</td>
		</tr>
		<tr>
			<td>Heating elements</td>
			<td>Won Brothers</td>
			<td>HT 300 Titanium</td>
			<td>Titanium rods placed at the bottom of tank</td>
		</tr>
		<tr>
			<td>Imaging array</td>
			<td>Verasonics</td>
			<td>L11-5v</td>
			<td>128 element with sensitivity from -55 to -49 dB</td>
		</tr>
		<tr>
			<td>Low gelling agarose</td>
			<td>Millipore Sigma</td>
			<td>A9414</td>
			<td></td>
		</tr>
		<tr>
			<td>Model vessel</td>
			<td>McMaster-Carr</td>
			<td>5234K98</td>
			<td>6.6 cm length, 0.6 cm inner diameter, 1 mm thickness</td>
		</tr>
		<tr>
			<td>Nanopure water</td>
			<td>Barnstead</td>
			<td>Nanopure Diamond</td>
			<td>ASTM type I, 18 Mohm-cm resistivity</td>
		</tr>
		<tr>
			<td>Plasma</td>
			<td>Vitalant</td>
			<td>4PF000</td>
			<td>Plasma frozen within 24 hours</td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>Biotek</td>
			<td>Synergy Neo HST Plate Reader</td>
			<td>For haemoglobin quantification</td>
		</tr>
		<tr>
			<td>Probe cover</td>
			<td>Civco</td>
			<td>610-362</td>
			<td></td>
		</tr>
		<tr>
			<td>Programming platform</td>
			<td>MATLAB (the Mathworks, Natick, MA, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant tissue-type plasminogen activator (rt-PA)</td>
			<td>Genentech</td>
			<td>Activase</td>
			<td></td>
		</tr>
		<tr>
			<td>Reservoir</td>
			<td>Cole-Parmer</td>
			<td>EW-07945-43</td>
			<td>60 mL capacity</td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Cole-Parmer</td>
			<td>EW-74900-20</td>
			<td>pump attached to the syringe to draw the flow in the flow channel at a pre-determined fized rate</td>
		</tr>
		<tr>
			<td>Transducer</td>
			<td>In-house customized</td>
			<td></td>
			<td>Eight-element, elliptically-focused transducer (9 cm major axis, 7 cm minor axis and 6 cm focal length), powered by custom designed and built class D amplifier and matching network</td>
		</tr>
		<tr>
			<td>Ultrasound scaning system</td>
			<td>Verasonics</td>
			<td></td>
			<td>Vantage Research Ultrasound System</td>
		</tr>
		<tr>
			<td>Water tank</td>
			<td>Advanced acrylics</td>
			<td>C133</td>
			<td>14 x 14 x 12, 1/2&#34;</td>
		</tr>
	</tbody>
</table>

an in vitro system to gauge the thrombolytic efficacy of histotripsy and a lytic drug

Deep vein thrombosis (DVT) is a global health concern. The primary approach to achieve vessel recanalization for critical obstructions is catheter-directed thrombolytics (CDT). To mitigate caustic side effects and the long treatment time associated with CDT, adjuvant and alternative approaches are under development. One such approach is histotripsy, a focused ultrasound therapy to ablate tissue via bubble cloud nucleation. Pre-clinical studies have demonstrated strong synergy between histotripsy and thrombolytics for clot degradation. This report outlines a benchtop method to assess the efficacy of histotripsy-aided thrombolytic therapy, or lysotripsy.
Clots manufactured from fresh human venous blood were introduced into a flow channel whose dimensions and acousto-mechanical properties mimic an iliofemoral vein. The channel was perfused with plasma and the lytic&#160;recombinant tissue-type plasminogen activator. Bubble clouds were generated in the clot with a focused ultrasound source designed for the treatment of femoral venous clots. Motorized positioners were used to translate the source focus along the clot length. At each insonation location, acoustic emissions from the bubble cloud were passively recorded, and beamformed to generate passive cavitation images. Metrics to gauge treatment efficacy included clot mass loss (overall treatment efficacy), and the concentrations of D-dimer (fibrinolysis) and hemoglobin (hemolysis) in the perfusate. There are limitations to this in vitro design, including lack of means to assess in vivo side effects or dynamic changes in flow rate as the clot lyses. Overall, the setup provides an effective method to assess the efficacy of histotripsy-based strategies to treat DVT.

The protocol outlined in this study highlights the details of venous clot modeling, lysotripsy for clot disruption, and ultrasound imaging in an in vitro setup of DVT. The adopted procedure demonstrates the steps necessary to assess clot disruption due to the combined effects of rt-PA and histotripsy bubble cloud activity. The benchtop setup was designed to mimic the characteristics of a venous iliofemoral vein. Figure 1A shows a model vessel that has the acoustic, mechanical, and geometrica...

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An In vitro System to Gauge the Thrombolytic Efficacy of Histotripsy and a Lytic Drug

Histotripsy-aided lytic delivery or lysotripsy is under development for the treatment of deep vein thrombosis. An in vitro procedure is presented here to assess the efficacy of this combination therapy. Key protocols for the clot model, image guidance, and assessment of treatment efficacy are discussed.

Deep vein thrombosis (DVT) is a global health concern. The primary approach to achieve vessel recanalization for critical obstructions is ...

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2.6K Views.  University of Chicago. Histotripsy-aided lytic delivery or lysotripsy is under development for the treatment of deep vein thrombosis. An in vitro procedure is presented here to assess the efficacy of this combination therapy. Key protocols for the clot model, image guidance, and assessment of treatment efficacy are discussed.

Video: An In vitro System to Gauge the Thrombolytic Efficacy of Histotripsy and a Lytic Drug

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

An In Vitro Enzymatic Assay to Measure Transcription Inhibition by Gallium(III) and H3 5,10,15-tris(pentafluorophenyl)corroles

Chemotherapy often involves broad-spectrum cytotoxic agents with many side effects and limited targeting. Corroles are a class of tetrapyrrolic macrocycles that exhibit differential cytostatic and cytotoxic properties in specific cell lines, depending on the identities of the chelated metal and functional groups. The unique behavior of functionalized corroles towards specific cell lines introduces the possibility of targeted chemotherapy.
Many anticancer drugs are evaluated by their ability to inhibit RNA transcription. Here we present a step-by-step protocol for RNA transcription in the presence of known and potential inhibitors. The evaluation of the RNA products of the transcription reaction by gel electrophoresis and UV-Vis spectroscopy provides information on inhibitive properties of potential anticancer drug candidates and, with modifications to the assay, more about their mechanism of action.
Little is known about the molecular mechanism of action of corrole cytotoxicity. In this experiment, we consider two corrole compounds: gallium(III) 5,10,15-(tris)pentafluorophenylcorrole (Ga(tpfc)) and freebase analogue 5,10,15-(tris)pentafluorophenylcorrole (tpfc). An RNA transcription assay was used to examine the inhibitive properties of the corroles. Five transcription reactions were prepared: DNA treated with Actinomycin D, triptolide, Ga(tpfc), tpfc at a [complex]:[template DNA base] ratio of 0.01, respectively, and an untreated control.
The transcription reactions were analyzed after 4 hr using agarose gel electrophoresis and UV-Vis spectroscopy. There is clear inhibition by Ga(tpfc), Actinomycin D, and triptolide.
This RNA transcription assay can be modified to provide more mechanistic detail by varying the concentrations of the anticancer complex, DNA, or polymerase enzyme, or by incubating the DNA or polymerase with the complexes prior to RNA transcription; these modifications would differentiate between an inhibition mechanism involving the DNA or the enzyme. Adding the complex after RNA transcription can be used to test whether the complexes degrade or hydrolyze the RNA. This assay can also be used to study additional anticancer candidates.

An In Vitro Enzymatic Assay to Measure Transcription Inhibition by GalliumIII and H3 5,10,15-trispentafluorophenylcorroles

Gallium(III) 5,10,15-(tris)pentafluorophenylcorrole and its freebase analogue exhibit low micromolar cell cytotoxicity. This manuscript describes an RNA transcription reaction, imaging RNA with an ethidium bromide-stained gel, and quantifying RNA with UV-Vis spectroscopy, in order to assess transcription inhibition by corroles and demonstrates a straightforward method of evaluating anticancer candidate properties.

Chemotherapy often involves broad-spectrum cytotoxic agents with many side effects and limited targeting. Corroles are a class of tetrapyrrolic ...

An Injectable and Drug-loaded Supramolecular Hydrogel for Local Catheter Injection into the Pig Heart

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the limited access to donor hearts. An example of a treatment to recover the function of the heart consists of the local delivery of drugs and bioactives from a hydrogel. In this paper a method is introduced to formulate and inject a drug-loaded hydrogel non-invasively and side-specific into the pig heart using a long, flexible catheter. The use of 3-D electromechanical mapping and injection via a catheter allows side-specific treatment of the myocardium. To provide a hydrogel compatible with this catheter, a supramolecular hydrogel is used because of the convenient switching from a gel to a solution state using environmental triggers. At basic pH this ureido-pyrimidinone modified poly(ethylene glycol) acts as a Newtonian fluid which can be easily injected, but at physiological pH the solution rapidly switches into a gel. These mild switching conditions allow for the incorporation of bioactive drugs and bioactive species, such as growth factors and exosomes as we present here in both in vitro and in vivo experiments. The in vitro experiments give an on forehand indication of the gel stability and drug release, which allows for tuning of the gel and release properties before the subsequent application in vivo. This combination allows for the optimal tuning of the gel to the used bioactive compounds and species, and the injection system.

Supramolecular hydrogelators based on ureido-pyrimidinones allow full control over the macroscopic gel properties and the sol&#8211;gel switching behavior using pH. Here, we present a protocol for formulating and injecting such a supramolecular hydrogelator via a catheter delivery system for local delivery directly in relevant areas in the pig heart.

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the ...

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected within in vivo oral biofilms. Furthermore, a system that uses natural human saliva as the nutrient source, instead of artificial media, is particularly desirable in order to support the expression of cellular and biofilm-specific properties that mimic the in vivo communities. We describe a method for the development of multi-species oral biofilms that are comparable, with respect to species composition, to supragingival dental plaque, under conditions similar to the human oral cavity. Specifically, this methods article will describe how a commercially available microfluidic system can be adapted to facilitate the development of multi-species oral biofilms derived from and grown within pooled saliva. Furthermore, a description of how the system can be used in conjunction with a confocal laser scanning microscope to generate 3-D biofilm reconstructions for architectural and viability analyses will be presented. Given the broad diversity of microorganisms that grow within biofilms in the microfluidic system (including Streptococcus, Neisseria, Veillonella, Gemella, and Porphyromonas), a protocol will also be presented describing how to harvest the biofilm cells for further subculture&#160;or DNA extraction and analysis. The limits of both the microfluidic biofilm system and the current state-of-the-art data analyses will be addressed. Ultimately, it is envisioned that this article will provide a baseline technique that will improve the study of oral biofilms and aid in the development of additional technologies that can be integrated with the microfluidic platform.

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque.&#160;Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted.

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

Robotic Production of Cancer Cell Spheroids with an Aqueous Two-phase System for Drug Testing

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing both mono-culture and co-culture spheroids in a high throughput 96-well plate format is described in this work. This approach utilizes an aqueous two-phase system to confine cells into a drop of the denser aqueous phase immersed within the second aqueous phase. The drop rests on the well surface and keeps cells in close proximity to form a single spheroid. This technology has been adapted to a robotic liquid handler to produce size-controlled spheroids and expedite the process of spheroid production for compound screening applications. Spheroids treated with a clinically-used drug show reduced cell viability with increase in the drug dose. The use of a standard micro-well plate for spheroid generation makes it straightforward to analyze viability of cancer cells of drug-treated spheroids with a micro-plate reader. This technology is straightforward to implement both robotically and with other liquid handling tools such as manual pipettes.

A protocol for robotic printing of cancer cell spheroids in a high throughput 96-well plate format using an aqueous two-phase system is presented.

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...