This work was supported by the Applied and Engineering Sciences (TTW) (Vidi-project 17543), part of NWO. The authors would like to thank Robert Beurskens, Luxi Wei, and Reza Pakdaman Zangabad from the Department of Biomedical Engineering and Michiel Manten and Geert Springeling from the Department of Experimental Medical Instrumentation for technical assistance, all from the Erasmus MC University Medical Center Rotterdam, the Netherlands.

The authors have nothing to disclose.

This protocol describes three methods on how to obtain and use 5 to 8-day old chicken embryos and their CAM as an in vivo&#160;model to study contrast-enhanced ultrasound imaging and microbubble-mediated drug delivery. The most critical steps for taking 5-day old (section 1.2) and 6 to 7-day old (section 1.3) embryos out of the shell are: 1) make the small hole in the top of the egg to go through the entire eggshell into the air sac before withdrawing egg white; 2) create smooth edges for the large opening in the shell. For the method for taking 8-day old embryos out of the shell (section 1.4) the most critical steps are: 1) Make a sufficient number of indents to create a nice crack along the egg; 2) Keep the egg submerged in PBS. To ensure embryo viability for all methods it is important to keep the egg and its contents at 37 &#176;C. In addition, avoid injecting into a CAM artery. Visually monitoring the heart rate of the embryo during the studies is recommended to ensure embryo vitality. To confirm the exact developmental stage of the embryo, the indication of Hamburger &#38; Hamilton22&#160;can be used.
It is important to prevent damage to the embryo, CAM, and yolk sack. This damage can affect the viability, blood flow, and visibility of the embryo and CAM. In addition, damage to the yolk sack and consequently a low rigidity of the membrane makes an injection into the CAM vessels impossible. A 5-day old embryo has a relatively small air sac so to be able to make a sufficiently large hole in the shell through which the egg content can be removed, 2 mL of egg white needs to be withdrawn. As a result, more space between the eggshell and embryo is created. After withdrawal of the egg white, a piece of tape needs to close off the hole where the needle went in. If egg white still leaks out, apply another piece of tape. Beside this, the application of tape on the hole on the side creates a vacuum inside the egg which prevents the egg content from falling out due to its own weight when the large hole is created in step 1.2.2.8. Damage to the embryo or CAM can also occur when the edge of the eggshell was too sharp or when the egg content is dropped into the weighing boat too rigorously, so the eggshell should be kept very close to the weighing boat. Between day 5 and 6 of development, the CAM starts to attach to the shell membrane32. This attachment increases the risk of damaging the embryo and CAM when taking the egg content out of the eggshell. By opening the egg after injection of PBS into it for a 6 to 7-day incubated egg or in a PBS-filled container as described for an 8-day incubated egg, the risk of damage is reduced. Regarding an injection into a CAM vein: if the first injection fails, a second injection can be done further upstream in the same vein if the damage was minor or in another CAM vein. Separation of the embryo and CAM from the yolk makes the embryo and CAM vessels optically transparent. As a consequence, the embryo loses its primary source of nutrients33. This loss of nutrients could be an explanation for the observed lower heart rate of 80 bpm in comparison to ~190 for a 6-day old embryo that is still in contact with the yolk30&#160;and the reduced survival time of 2 h after this separation procedure. Another factor that can play a role in the reduced heart rate and survival time is the challenge to keep the yolk-separated embryo and CAM vessels at 37 &#176;C. A microscope stage incubator may be of aid. In addition to this, the detachment of the CAM from the yolk likely leads to mechanical changes in the tissue since the membrane tension becomes less. The lower membrane tension may cause an increased inner vessel shear rate which leads to a lower heart rate.
The ex ovo chicken embryo and CAM vessels have some limitations as in vivo model, including short time observations only, for contrast enhanced ultrasound imaging and microbubble-mediated drug delivery studies. Due to the small blood volume of 100&#177;23 &#181;L at day 5 and 171&#177;23 &#181;L at day 634, a maximum volume of ~5 &#181;L can be injected. In the later stages of development (day 7 and older), the vessel stiffness increases and the yolk elasticity decreases. This can complicate a successful injection in older embryos. Once the microbubbles are injected, they circulate for hours because the chicken embryo does not have a fully developed immune system at this stage35. Therefore, microbubbles are not cleared within ~6 min as in humans36,37&#160;making typical ultrasound molecular imaging studies with a 5-10 min waiting period for non-bound targeted microbubbles to be cleared38&#160;not feasible. In order to target microbubbles, suitable ligands able to bind to avian endothelial cells need to be used such as previously described for the angiogenesis marker &#945;v&#946;37. Other aspects to consider for this model are the increased difficulty of separating the embryo and CAM vessels from the yolk in older embryos (&#62; 8 days) and lower hematocrit of ~20%39&#160;in comparison to humans. The latter may affect microbubbles oscillations because it is known that microbubble oscillations are damped in a more viscous environment40. CAM arteries are less oxygenated than CAM veins41,42. This difference should be taken into account when for example studying photoacoustic imaging of blood oxygenation.
The methods described here allow the egg content to be taken out of the eggshell on the day of the ultrasound imaging or drug delivery study, typically at day 5 to 8 of incubation. This is different to existing methods where the egg content is taken out of the shell after a 3-day incubation and further developed as ex ovo&#160;culture18,20,21. The advantages are a higher survival rate of 90% for 5-day, 75% for 6-day, 50% for 7-day, and 60% for 8-day old incubated eggs in comparison to ~50% for 3-day old embryos taken out of the eggshell and further incubated ex ovo1,18&#160;the avoidance of antibiotics during culture18,20&#160;and large sterile incubator for the ex ovo culture. The survival of the 6-to-8-day old embryos is lower because the CAM starts to attach itself to the shell21&#160;which leaves the CAM membrane more prone to rupture upon extraction. The separation of the embryo with the CAM form the yolk is also described making the embryo and CAM optically transparent.
By placing the egg content in different setups, the chicken embryo and CAM can be used for a multitude of ultrasound imaging studies, like IVUS, photoacoustic, without or with ultrasound contrast agents in 2D and 3D. The focus can be on developing new ultrasound pulsing schemes or testing out novel transducers. Besides this, the model can also be used to investigate novel ultrasound contrast agents and their behavior in blood vessels under flow. Since the mechanism of microbubble-mediated drug delivery is still unknown43, the use of the in vivo CAM model may help in elucidating the mechanism by studying the microbubble behavior in relation to the cellular response. Finally, the CAM vessels have proven to be a suitable system to investigate xenograft tumor transplantation44. This creates the possibility to use the CAM vessel as a model to investigate tumor imaging using ultrasound and to investigate the blood flow inside the tumor using CEUS. The tumors are typically grafted on the CAM vessels of 8 or 9-day old embryos1,14,45, for which the embryo&#160;is taken out of the eggshell at day 3 of incubation and further developed ex ovo. The methods described in this protocol could be used to grow embryos in ovo until the day of tumor&#160;grafting.
The authors trust that this paper will be helpful for researchers that want to use chicken embryos and their chorioallantoic membrane (CAM) as an in vivo&#160;model for applications of contrast agents and flow studies.

Ex ovo chicken embryos and the blood-vessel rich chorioallantoic membrane (CAM) have proven to be a suitable model to investigate various biological and biomedical processes like embryogenesis, oncology, and drug delivery1,2,3,4. Ultrasound has been used for imaging of the embryonic heart development4,5&#160;and for activating cavitation nuclei upon injection, such as microbubbles, for vascular drug delivery6,7. Chicken embryos are inexpensive, require less infrastructure and equipment, and have less strict legislation compared to other animal models8. The chicken embryo and CAM vessels are easily accessible after opening the egg whereas this proves to be much more difficult with mammalian embryos and vessels. Besides this, the chicken embryo and CAM vessels provide a heartbeat and a pulsating blood flow. The CAM shows similarities in vessel anatomy with mammals and can be used for drug screening8,9,10. Because of these characteristics, the CAM vessels have also proven to be a suitable model to investigate contrast enhanced ultrasound imaging (CEUS)11,12,13,14,15,16. In addition, the model can be used to optically investigate the behavior of ultrasound contrast agents in an ultrasound field using an ultra-high-speed camera and the effect of acoustic radiation force on propelling, binding and extravasation of drugs7,17,18,19. Although the chicken embryo and CAM are less suitable for long term experiments, they can be beneficial for short term in vivo experiments.
To increase visibility and controllability over the chicken embryo and CAM during experiments, it is important to take the egg content containing the embryo and CAM out of the eggshell18. Previous chicken embryo studies involving ultrasound contrast agents used 5 to 6-day old embryos7,11,12,17,19&#160;and 14 to 18-day old embryos13,14,15,16. Multiple approaches have been described in detail to take the egg content out of the shell18,20,21. However, to the best of our knowledge, the previously published approaches focus on taking the egg content out of the eggshell after 3 days of incubation (i.e., Hamburger &#38; Hamilton (HH) stage 19-2022), and continue the culture ex ovo. This ex ovo culture approach has multiple disadvantages including increased risk of fatalities during culture (~50%)1,18, the use of antibiotics18,20, and decreased total vessel length in comparison to in ovo growth23. Since culturing the embryo within the eggshell is providing the most natural environment, it is easiest to incubate the embryo within the eggshell until the day of the experiment. For this reason, an approach in which the egg content is taken out of the eggshell at 5 to 8 days of incubation would be beneficial especially for experiments on 5 to 8-day old embryos.
In this protocol, we describe three methods to take the egg content out of the eggshell when the embryo is at day 5 to 8 of development (HH 26-3522) allowing the embryo to develop within the eggshell until the day of the experiment. The CAM vessel size ranges from 10-15 &#181;m in diameter, in the smaller capillaries of an 8-day old embryo24,&#160;to 115-136 &#181;m in diameter in the larger vessel of 6 and 8-day old embryos24,25. The three described methods only require basic lab tools and reduce the risk of complications before the experiment has begun, thereby reducing unnecessary costs and labor. We also detail a method to separate the membrane containing the embryo and CAM from the yolk sack making the CAM optically transparent for microscopy studies. Because the membrane containing the embryo and CAM can be pinned down on for example a holder with an acoustic membrane, the setup can also be made acoustically transparent26, allowing the combination of microscopy and ultrasound studies when the light path will be affected by the yolk. Finally, we describe several other ultrasound setups that can be used for ultrasound or CEUS imaging.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A9539</td>
			<td></td>
		</tr>
		<tr>
			<td>Clamp (Kocher clamp)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cling film</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Holder with acoustic membrane (CLINIcell 25 cm2)</td>
			<td>MABIO, Tourcoing, France</td>
			<td>CLINIcell25-50-T FER 00106</td>
			<td></td>
		</tr>
		<tr>
			<td>Demi water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable plastic Pasteur pipets</td>
			<td>VWR</td>
			<td>612-1747</td>
			<td></td>
		</tr>
		<tr>
			<td>Eggs</td>
			<td colspan="2">Drost Pluimveebedrijf Loenen BV, the Netherlands</td>
			<td>Freshly fertilized</td>
		</tr>
		<tr>
			<td>Fridge 15 &#176;C</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass capillary needles</td>
			<td>Drummond</td>
			<td>1-000-1000</td>
			<td>Inside diameter: 0.0413 inch</td>
		</tr>
		<tr>
			<td>Heating plate 37 &#176;C</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Humidified incubator 37 &#176;C</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Insect specimen pins</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Metal egg holder</td>
			<td></td>
			<td></td>
			<td>Custom made by Experimental Medical Instrumentation at Erasmus MC. See figure 1 A,B</td>
		</tr>
		<tr>
			<td>Metal weighing boat holder</td>
			<td></td>
			<td></td>
			<td>Custom made by Experimental Medical Instrumentation at Erasmus MC. See figure 1 C,D</td>
		</tr>
		<tr>
			<td>Microinjection system</td>
			<td>FUJIFILM VisualSonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Sigma-Aldrich</td>
			<td>M8410-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle, 19 G</td>
			<td>VWR (TERUMO)</td>
			<td>613-5392</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-bufferes saline (PBS), 1x</td>
			<td>ThermoFisher</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish, 1 L</td>
			<td></td>
			<td></td>
			<td>Glass</td>
		</tr>
		<tr>
			<td>Petri dish, 90 mm diameter</td>
			<td>VWR</td>
			<td>391-0559</td>
			<td></td>
		</tr>
		<tr>
			<td>Preclinical animal ultrasound machine (Vevo 2100)</td>
			<td>FUJIFILM VisualSonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Probe (MS250)</td>
			<td>FUJIFILM VisualSonics</td>
			<td></td>
			<td>30 MHz transmit and 15 MHz receive frequency</td>
		</tr>
		<tr>
			<td>Probe (MS550s)</td>
			<td>FUJIFILM VisualSonics</td>
			<td></td>
			<td>transmission frequency of 40 MHz</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>VWR (SWANN-MORTON)</td>
			<td>233-5363</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors, small</td>
			<td>Fine Science Tools (FST)</td>
			<td>14558-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe, 5 mL</td>
			<td>VWR (TERUMO)</td>
			<td>613-0973</td>
			<td></td>
		</tr>
		<tr>
			<td>Table spoon</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tape (Scotch Magic tape)</td>
			<td>Scotch</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue paper</td>
			<td>Tork</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers large&#160;</td>
			<td>VWR (USBECK Laborger&#228;te)</td>
			<td>232-0107</td>
			<td>See figure 1E</td>
		</tr>
		<tr>
			<td>Tweezers small</td>
			<td>DUMONT Medical, Switzerland</td>
			<td>0103-5/45</td>
			<td>See figure 1F</td>
		</tr>
		<tr>
			<td>Ultrasound contrast agent (custum made F-type)</td>
			<td></td>
			<td></td>
			<td>Produced as described by: Daeichin, V. et al. Microbubble Composition and Preparation for Imaging : In Vitro and In Vivo Evaluation. IEEE TRANSACTIONS ON ULTRASONICS. 64 (3), 555&#8211;567 (2017).</td>
		</tr>
		<tr>
			<td>Ultrasound contrast agent (MicroMarker)</td>
			<td>FUJIFILM VisualSonics, Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound contrast agent (Definity)</td>
			<td colspan="2">Lantheus medical imaging, United States</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound gel</td>
			<td>Aquasonic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Waxi film (Parafilm)</td>
			<td>Parafilm</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Weighing boats (85 &#215; 85 &#215; 24 mm)</td>
			<td>VWR</td>
			<td>611-0094</td>
			<td></td>
		</tr>
	</tbody>
</table>

the preparation of chicken ex ovo embryos and chorioallantoic membrane vessels as in vivo model for contrast-enhanced ultrasound imaging and microbubble-mediated drug delivery studies

The chicken embryo and the blood-vessel rich chorioallantoic membrane (CAM) is a valuable in vivo model to investigate biomedical processes, new ultrasound pulsing schemes, or novel transducers for contrast-enhanced ultrasound imaging and microbubble-mediated drug delivery. The reasons for this are the accessibility of the embryo and vessel network of the CAM as well as the low costs of the model. An important step to get access to the embryo and CAM vessels is to take the egg content out of the eggshell. In this protocol, three methods for taking the content out of the eggshell between day 5 and&#160;8 of incubation are described thus allowing the embryos to develop inside the eggshell up to these days. The described methods only require simple tools and equipment and yield a higher survival success rate of 90% for 5-day, 75% for 6-day, 50% for 7-day, and 60% for 8-day old incubated eggs in comparison to ex ovo cultured embryos (~50%). The protocol also describes how to inject cavitation nuclei, such as microbubbles, into the CAM vascular system, how to separate the membrane containing the embryo and CAM from the rest of the egg content for optically transparent studies, and how to use the chicken embryo and CAM in a variety of short-term ultrasound experiments. The in vivo&#160;chicken embryo and CAM model is extremely relevant to investigate novel imaging protocols, ultrasound contrast agents, and ultrasound pulsing schemes for contrast-enhanced ultrasound imaging, and to unravel the mechanisms of ultrasound-mediated drug delivery.

In this protocol, we describe three methods to take the egg content out of the shell at day 5-8 of incubation (HH 26-3522). Figure 6&#160;shows the egg content in weighing boats after it was taken out of the shell. The 5-day old embryo and CAM (Figure 6A) was taken out using the method described in section 1.2. The 6 and 7-day old embryos and CAM (Figure 6B,C) were taken out using the method described in section 1.3. The 8-day old embryo and CAM (Figure 6D) was taken out using the method described in section 1.4. No bleeding or damage to the embryo or CAM can be observed, indicating that these methods can be used to safely get the egg content out of the shell without harming the embryo or the CAM vessels. When executed correctly, the method for the 5-day old embryos will provide a viable embryo and intact CAM in 90% of all procedures. The viability rate is based on the total number of fertilized eggs successfully extracted from the eggshell.&#160;With the second method, for 6 and 7-day incubated eggs, the chance of a viable embryo and intact CAM is around 75% for 6-day old and around 50% for 7-day old. With the third method described for 8-day old embryos, the chance of a viable embryo and intact CAM is around 60%. Differences in developmental stages between the 5 and 8-day old embryos can be observed which concurs with Hamburger and Hamilton22. Both the size of the embryo and the complexity of the CAM vessels increase during development (Figure 6A-D). Figure 6C shows a thin patch of agarose on top of the egg content that allows the embryo and CAM to be imaged using the ultrasound setup shown in Figure 5C. After the egg content is taken out of the shell the heartbeat of the embryo is visible with the naked eye. The heart rate of these ex ovo&#160;embryos is similar to in ovo embryos at 183 beats per minute (bpm) at day 5 up to ~208 bpm at day 830. When kept humidified and at 37 &#176;C, the embryo will maintain this heart rate for ~5 h in the experimental ultrasound setups.
Multiple complications can occur during the previously described three methods. Figure 7A shows trapped air under the CAM which makes the embryo unsuitable for ultrasound imaging and the pressure of the air bubble(s) can also damage the embryo and/or CAM. This problem arises when the air sac inside the shell does not make contact with the air outside the shell when taking the egg content out of the shell. Figure 7B shows a small leakage of yolk from the yolk sack in the top right of the image. This can occur while taking the egg content out of the shell when the yolk sack gets damaged by sharp edges of the shell or when the yolk sack is penetrated by the tweezers. Leakage of the yolk can affect the visibility of the embryo and the CAM vessels. Figure 7C shows an embryo in which an air bubble is trapped under the CAM. This sometimes occurs in the embryonic development. Another complication which can occur is damage to the vessels. This damage can be created while taking the egg content out of the shell or when performing an injection (Figure 7D). Besides this, the embryo and vessels can also dry out over time (Figure 7E). This occurs when the egg content is not sprinkled with PBS. The drying out of the embryo can result in massive capillary obstructions (Figure 7F) which affects the viability of the embryo. The massive capillary obstructions can also occur during development or when the heartbeat of the embryo is not stable.
After the egg content is taken out of the shell without any complication, the embryo can be injected with, for example, ultrasound contrast agents such as microbubbles (Figure 3C). Figure 8 shows circulating microbubbles in the lumen of the blood vessel upon injection. These microbubbles are carried along with the blood flow and stay present in the blood circulation for several hours (Supplemental Video 1). The presence of these microbubbles in the circulation creates the possibility to perform different types of CEUS and drug delivery experiments7,11,12. The CAM is ideal to investigate novel ultrasound contrast detection methods for which we show three examples. Figure 9A shows high frequency ultrasound subharmonic imaging of a 6-day old chicken embryo in B-Mode and CEUS before and after microbubble injection. Here, the CAM vessels were injected with 5 &#181;L of ultrasound contrast agent and imaging was performed with a preclinical animal ultrasound machine with a MS250 probe (30 MHz transmit and 15 MHz receive frequency, 10% power). Before microbubble injection, contrast can already be seen inside the embryonic heart in the B-Mode images (Figure 9A-I). This phenomenon is due to the presence of a nucleus in the avian red blood cell which increases the contrast of blood in ultrasound imaging5,31. The addition of the microbubbles increased the contrast and the visibility of the embryo, both in the B-Mode and CEUS imaging. Figure 9B shows an optical and a high frequency 3D subharmonic image of a 6-day old embryo and the surrounding vessels. The CAM was injected with 5 &#181;L of ultrasound contrast agent and imaging was performed with a preclinical animal ultrasound machine with MS550s probe (transmission frequency of 40 MHz, peak negative pressure ~300 kPa). These results show that the CEUS imaging combined with a contrast agent can also be used to create high frequency 3D subharmonic images and to image the blood vessels outside the embryo. Figure 9C shows an optical image and an ultraharmonic intravascular ultrasound (IVUS) image made with a custom probe of CAM microvessels of a 6-day old embryo (26 MHz transmit and 39 and 65 MHz receive frequency). CAM vessels were injected with 4 &#177; 1 &#181;L ultrasound contrast agent. The optical image and IVUS image are from the same embryo and same region of interest showing corresponding vessel networks.
The chicken embryo and CAM vessels can also be used to investigate ultrasound-mediated drug delivery for which we show one example. Since the yolk obstructs the light path during imaging, the removal of the yolk sack is necessary to optically investigate drug delivery in the embryo and CAM vessels. For this study, the embryo and CAM were prepared for microscopic imaging as explained in section 2.2 by separating the membrane containing the embryo and CAM from the yolk sack (Figure 4C). In these embryos, the heart rate is stable around 80 bpm and the embryos stay alive for up to 2 h when kept at a 37 &#176;C7. Figure 10&#160;shows an ultrasound and microbubble mediated drug delivery study in endothelial cells of the CAM vessels. Lipid-coated microbubbles, targeted to the vessel wall using &#945;v&#946;3-antibodies and stained with the fluorescent dye DiI7, were injected into the CAM vessels (Figure 10A,C). CAM vessel endothelial cell nuclei were stained with Hoechst 33342 (Figure 10B) and the model drug Propidium Iodide (PI) was used to visualize sonoporation7. Both these dyes were injected simultaneously with the microbubbles. Upon ultrasound treatment (1 MHz, 200 kPa peak negative pressure, single burst of 1000 cycles), PI uptake was observed in the nuclei closest to the targeted microbubbles (Figure 10D). This shows that the ultrasound-induced oscillations of the targeted microbubbles were able to create a pore in the endothelial cell membrane.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62076/62076fig01.jpg" /> 
Figure 1. Embryo preparation equipment. (A-B) top and side view of the metal egg holder and (C-D) top and side view of the metal weighing boat holder. (E-F) tweezers needed to take the egg content out of the shell. Scale in cm. <a href="https://www.jove.com/files/ftp_upload/62076/62076fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62076/62076fig02.jpg" /> 
Figure 2. Embryo removal procedure. (A) Small indent on top of the egg, indicated by the black circle. (B) Small indent 2/3 down the egg, indicated by the black circle. (C) Withdrawing ~2 mL of egg white. (D) Sealed gap on the side with tape. (E) Enlarging the small opening on the top of the egg. (F) The embryo becomes visible after removing part of the shell. (G-H) After rotating the egg 180&#176;, the embryo floats up and will become invisible (arrows indicate moving direction of the embryo). After 1-2 min, the embryo is invisible from the bottom. (I) After scratching the membrane, the egg content drops into the weighing boat.&#160;<a href="https://www.jove.com/files/ftp_upload/62076/62076fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62076/62076fig03.jpg" /> 
Figure 3. Injection of microbubbles into the CAM vessels. (A) Glass capillary needle. Scale in cm. (B) Propidium iodide (PI) solution (left drop) and microbubbles (right drop) prior to aspiration before injection. Needle (outlined in black) can be seen in the top right corner (C) Microbubble injection. The capillary needle tip is positioned inside the lumen of one of the veins (left). Microbubbles, the white cloud indicated with an arrow, are injected and disperse following the blood stream (right). Scale bar represents 1 mm.&#160;<a href="https://www.jove.com/files/ftp_upload/62076/62076fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62076/62076fig04.jpg" /> 
Figure 4. Removing embryo and CAM from yolk sack and placing on holder with acoustic membrane. (A) Holder with acoustic membrane filled with agarose layer. (B) Chicken embryo and CAM vessel in weighing boat before cutting. Dotted line indicates the cutting line around the CAM. (C) Chicken embryo and CAM separated from yolk and pinned down on acoustic membrane. (D) Pinned down chicken embryo with an acoustically and optically transparent membrane in a holder (blue) placed on top of the CAM. The holder can be filled with demi water so a water dipping objective can be used. All scale bars represent 1 cm.&#160;<a href="https://www.jove.com/files/ftp_upload/62076/62076fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62076/62076fig05.jpg" /> 
Figure 5. Different setups for chicken embryo and CAM ultrasound imaging. (A) Setup for ultrasound imaging from the side. Chicken embryo was placed in a custom-adjusted weighing boat with one acoustically transparent wall and placed in a 37 &#176;C water bath. The ultrasound transducer was positioned on the left (a) side next to the acoustically transparent wall and the laser (b) for photoacoustic imaging on top. (B) Setup for ultrasound imaging from the top. Embryo and CAM were submerged in a beaker of PBS that was placed in a 37 &#176;C water bath. Dashed outline shows the 2 L glass beaker (a) with the 500 mL glass beaker (b) inside. (C) Setup for ultrasound imaging from the top with a movable transducer. A thin agarose pad (dotted line) was placed on top of the embryo with a thin layer of PBS as coupling between the transducer and the agarose surface.&#160;<a href="https://www.jove.com/files/ftp_upload/62076/62076fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62076/62076fig06.jpg" /> 
Figure 6. Egg content outside the shell. (A) Egg content taken out of the shell after 5 days of incubation. The chorioallantoic membrane (CAM), embryo body (EB), anterior and posterior vitelline veins (*), and appropriate sites for injection (arrowheads) are indicated. (B) Egg content taken out of the shell after 6 days of incubation. The anterior and posterior vitelline veins (*) and appropriate sites for injection (arrowheads) are indicated. (C) Egg content taken out of the shell after 7 days of incubation. A patch of agarose is placed on top to allow for ultrasound imaging. The corners of the agarose patch are indicated with black circles. (D) Egg content taken out of the shell after 8 days of incubation. All scale bars represent 1 cm.&#160;<a href="https://www.jove.com/files/ftp_upload/62076/62076fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/62076/62076fig07.jpg" /> 
Figure 7. Complications which can occur during the procedures with the chicken embryo and CAM model. (A) Air bubbles trapped under the CAM when taking the egg content out of the shell using method 1.2 (5-day old embryo) or 1.3 (6&#160;to 7-day old embryo). (B) Small leakage of yolk indicated with an arrow on the top right (6-day old embryo). (C) Air trapped under the CAM, indicated by the black dotted circle (7-day old embryo). (D) Bleeding, indicated with the black arrows (5-day old embryo. (E) Dried out embryo and CAM (5-day old embryo). (F) Massive capillary obstructions (5-day old embryo). All scale bars represent 1 cm.&#160;<a href="https://www.jove.com/files/ftp_upload/62076/62076fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/62076/62076fig08.jpg" /> 
Figure 8. Microbubbles in CAM blood vessel. The vessel wall is indicated with a dotted line and single microbubbles are indicated with arrows. Scale bar represents 20 &#181;m. The corresponding microscopy recording can be found in Supplemental Video 1.&#160;<a href="https://www.jove.com/files/ftp_upload/62076/62076fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/62076/62076fig09.jpg" /> 
Figure 9. Contrast-enhanced ultrasound imaging in chicken embryos and CAM vessels. (A) Maximum-intensity projection of B-Mode (I, III) and real-time subharmonic (II, IV) images (preclinical animal ultrasound machine with MS250 probe, 30 MHz transmitting and 15 MHz receiving frequency, 10% power) of a 6-day old embryo with a patch of agarose on top. Top images (I, II) show results before and bottom (III, IV) after injection of 5 &#181;L ultrasound contrast agent. Scale bar represents 1 mm. This image has been modified with permission from Daeichin et al.&#160;201511&#160;(B) Optical (left) and 3D subharmonic imaging (right) of a 6-day old chicken embryo with a patch of agarose on top. CAM vessels were injected with 5 &#181;L ultrasound contrast agent and imaging was performed with a high-frequency probe (preclinical animal ultrasound machine with MS550s probe, transmission frequency of 40 MHz, peak negative pressure ~300 kPa, rendered in preclinical animal ultrasound machine 3-D mode). Scale bar represents 5 mm. This image has been modified with permission from Daeichin et al.&#160;201511. (C) Optical image (left) and mean intensity projection of ultraharmonic intravascular ultrasound (IVUS) (right) of the CAM microvasculature of a 6-day old embryo. CAM vessels were injected with 4 &#177; 1 &#181;L contrast agent. Ultraharmonic IVUS imaging was performed with a custom IVUS probe (transmission frequency 35 MHz, peak negative pressure 600 kPa). Both images are made from the same embryo and region of interest. Arrows indicate corresponding vessels in the two images. Scale bar represents 1 mm. This image has been modified with permission from Maresca et al. 201412.&#160;<a href="https://www.jove.com/files/ftp_upload/62076/62076fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/62076/62076fig10.jpg" /> 
Figure 10. Drug delivery to CAM vessel endothelial cells in 6-day old embryo.&#160;(A) Brightfield image of six &#945;v&#946;3-targeted microbubbles, indicated with white arrows, adhering to the vessel wall before ultrasound treatment. (B) Endothelial cell nuclei fluorescently stained before ultrasound treatment. (C) Fluorescent image of the stained targeted microbubbles, indicated with white arrows, before ultrasound treatment. (D) Uptake of the model drug propidium iodide (PI) into the cell nuclei underneath the targeted microbubbles after ultrasound treatment (1 MHz, 200 kPa peak negative pressure, single burst of 1000 cycles). Scale bar represents 10 &#181;m and applies to all images. This image has been modified with permission from Skachkov et al.&#160;20147.&#160;<a href="https://www.jove.com/files/ftp_upload/62076/62076fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
SUPPLEMENTARY FILES
Supplemental Video 1. Microbubbles in CAM blood vessel. Scale bar represents 20 &#181;m.&#160;<a href="https://www.jove.com/files/ftp_upload/62076/Supplemental Video 1.mp4" target="_blank">Please click here to download this video.</a>

Watch this Scientific Journal Video about The Preparation of Chicken Ex Ovo Embryos and Chorioallantoic Membrane Vessels as In Vivo Model for Contrast-Enhanced Ultrasound Imaging and Microbubble-Media

The Preparation of Chicken Ex Ovo Embryos and Chorioallantoic Membrane Vessels as In Vivo Model for Contrast-Enhanced Ultrasound Imaging and Microbubble-Mediated Drug Delivery Studies

This protocol describes three methods on how to obtain and use 5 to 8-day old chicken embryos and their chorioallantoic membrane (CAM) as an in vivo&#160;model to study contrast-enhanced ultrasound imaging and microbubble-mediated drug delivery.

The chicken embryo and the blood-vessel rich chorioallantoic membrane (CAM) is a valuable in vivo model to investigate biomedical processes, new ...

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4.1K Views.  Erasmus MC University Medical Center Rotterdam. This protocol describes three methods on how to obtain and use 5 to 8-day old chicken embryos and their chorioallantoic membrane (CAM) as an in vivo model to study contrast-enhanced ultrasound imaging and microbubble-mediated drug delivery.

Video: The Preparation of Chicken Ex Ovo Embryos and Chorioallantoic Membrane Vessels as In Vivo Model for Contrast-Enhanced Ultrasound Imaging and Microbubble-Mediated Drug Delivery Studies

Contrast Ultrasound Targeted Treatment of Gliomas in Mice via Drug-Bearing Nanoparticle Delivery and Microvascular Ablation

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the microvasculature are controlled by varying ultrasound pulsing parameters.  Specifically, we are testing whether such approaches may be used to treat malignant brain tumors through drug delivery and microvascular ablation.  Preliminary studies have been performed to determine whether targeted drug-bearing nanoparticle delivery can be facilitated by the ultrasound mediated destruction of "composite" delivery agents comprised of 100nm poly(lactide-co-glycolide) (PLAGA) nanoparticles that are adhered to albumin shelled microbubbles. We denote these agents as microbubble-nanoparticle composite agents (MNCAs). When targeted to subcutaneous C6 gliomas with ultrasound, we observed an immediate 4.6-fold increase in nanoparticle delivery in MNCA treated tumors over tumors treated with microbubbles co-administered with nanoparticles and a 8.5 fold increase over non-treated tumors. Furthermore, in many cancer applications, we believe it may be desirable to perform targeted drug delivery in conjunction with ablation of the tumor microcirculation, which will lead to tumor hypoxia and apoptosis. To this end, we have tested the efficacy of non-theramal cavitation-induced microvascular ablation, showing that this approach elicits tumor perfusion reduction, apoptosis, significant growth inhibition, and necrosis. Taken together, these results indicate that our ultrasound-targeted approach has the potential to increase therapeutic efficiency by creating tumor necrosis through microvascular ablation and/or simultaneously enhancing the drug payload in gliomas.

Insonation of microbubbles is a promising strategy for tumor ablation at reduced time-averaged acoustic powers, as well as for the targeted delivery of therapeutics. The purpose of the present study is to develop low duty cycle ultrasound pulsing strategies and nanocarriers to maximize non-thermal microvascular ablation and payload delivery to subcutaneous C6 gliomas.

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i)&#160;to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

This procedure demonstrates in vivo near IR fluorescence imaging of collagen remodeling activities in mice as well as ex vivo staining of collagens in tissue sections using caged collagen mimetic peptides that can be photo-triggered to hybridize with denatured collagen strands.

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an ...

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 ...

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential to establish an ex vivo model that can evaluate SIM performance accurately, for developing high-performance SIMs. In our previous study, we developed a new ex vivo model that can be used to evaluate the performance of various SIMs in detail by applying constant tension to the specimen&#39;s ends. We also confirmed that the proposed new ex vivo model allows accurate submucosal elevation height (SEH) measurement under uniform conditions and detailed comparisons of the performances of various types of SIMs. Here, we describe the new ex vivo model and explain the detailed setup methodology of this model. Since all parts of the new model were easy to obtain, the setup of the new model could be completed quickly. SEH of various SIMs could be measured more accurately by using the new model. The critical factor that determines SIM performance can be identified using the new model. SIM development speed will drastically increase after the factor has been identified.

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

We developed a new ex vivo model that applies constant tension to the porcine gastric specimen. This development made it possible to evaluate the performance (the height and duration of the submucosal elevation) of various SIMs accurately.&#160; The detailed setup methodology of this new model is explained.

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential ...

In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located in. Although traditional imaging techniques are useful in identifying protein interactions and morphology in fixed tissue, they are not able to give insight as to how quickly cells are able to bind and uptake proteins, and once activated how their morphology changes in vivo. In the present study, we ask 2 major questions: 1) what is the time-course of fibroblast activation via toll-like receptor-4 (TLR4) and lipopolysaccharide (LPS) interaction and 2) how do these cells behave once activated? Using 2-photon imaging, we have developed a novel technique to assess the ability of LPS-FITC to bind to its cognate receptor, TLR4, expressed on peripheral fibroblasts in the genetic reporter mouse line; FSP1cre; tdTomatolox-stop-lox&#160;in vivo. This unique approach allows researchers to create in-depth, time-lapse videos and/or pictures of proteins interacting with live cells that allows one to have an increased level of granularity in understanding how proteins can alter cellular behavior.

Morphological changes occur in immune responsive fibroblast cells following activation and promote alterations in cellular recruitment. Utilizing 2-photon imaging in conjunction with a genetically engineered Fibroblast-specific protein 1 (FSP1)-cre; tdTomato floxed-stop-floxed (TB/TB) mouse line and green fluorescently tagged lipopolysaccharide-FITC, we can illustrate highly specific uptake of lipopolysaccharide in dermal fibroblasts and morphological changes in vivo.

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located ...

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...

Contrast-Enhanced Subharmonic Aided Pressure Estimation (SHAPE) Using Ultrasound Imaging with a Focus on Identifying Portal Hypertension

Noninvasive, accurate measurement of pressures within the human body has long been an important but elusive clinical goal. Contrast agents for ultrasound imaging are gas-filled, encapsulated microbubbles (diameter &#60; 10 &#956;m) that traverse the entire vasculature and enhance signals by up to 30 dB. These microbubbles also produce nonlinear oscillations at frequencies ranging from the subharmonic (half of the transmit frequency) to higher harmonics. The subharmonic amplitude has an inverse linear relationship with the ambient hydrostatic pressure. Here an ultrasound system capable of performing real-time, subharmonic aided pressure estimation (SHAPE) is presented. During ultrasound contrast agent infusion, an algorithm for optimizing acoustic outputs is activated. Following this calibration, subharmonic microbubble signals (i.e., SHAPE) have the highest sensitivity to pressure changes and can be used to noninvasively quantify pressure. The utility of the SHAPE procedure for identifying portal hypertension in the liver is the emphasis here, but the technique has applicability across many clinical scenarios.

Contrast-Enhanced Subharmonic Aided Pressure Estimation SHAPE Using Ultrasound Imaging with a Focus on Identifying Portal Hypertension

A protocol for noninvasively estimating ambient pressures utilizing subharmonic ultrasound imaging of infused contrast microbubbles (following appropriate calibration) is described with examples from human patients with chronic liver disease.

Noninvasive, accurate measurement of pressures within the human body has long been an important but elusive clinical goal. Contrast agents for ultrasound ...

Multi-timescale Microscopy Methods for the Characterization of Fluorescently-labeled Microbubbles for Ultrasound-Triggered Drug Release

Microbubble contrast agents hold great promise for drug delivery applications with ultrasound. Encapsulating drugs in nanoparticles reduces systemic toxicity and increases circulation time of the drugs. In a novel approach to microbubble-assisted drug delivery, nanoparticles are incorporated in or on microbubble shells, enabling local and triggered release of the nanoparticle payload with ultrasound. A thorough understanding of the release mechanisms within the vast ultrasound parameter space is crucial for efficient and controlled release. This set of presented protocols is applicable to microbubbles with a shell containing a fluorescent label. Here, the focus is on microbubbles loaded with poly(2-ethyl-butyl cyanoacrylate) polymeric nanoparticles, doped with a modified Nile Red dye. The particles are fixed within a denatured casein shell. The microbubbles are produced by vigorous stirring, forming a dispersion of perfluoropropane gas in the liquid phase containing casein and nanoparticles, after which the microbubble shell self-assembles. A variety of microscopy techniques are needed to characterize the nanoparticle-stabilized microbubbles at all relevant timescales of the nanoparticle release process. Fluorescence of the nanoparticles enables confocal imaging of single microbubbles, revealing the particle distribution within the shell. In vitro ultra-high-speed imaging using bright-field microscopy at 10 million frames per second provides insight into the bubble dynamics in response to ultrasound insonation. Finally, nanoparticle release from the bubble shell is best visualized by means of fluorescence microscopy, performed at 500,000 frames per second. To characterize drug delivery in vivo, the triggered release of nanoparticles within the vasculature and their extravasation beyond the endothelial layer is studied using intravital microscopy in tumors implanted in dorsal skinfold window chambers, over a timescale of several minutes. The combination of these complementary characterization techniques provides unique insight into the behavior of microbubbles and their payload release at a range of time and length scales, both in vitro and in vivo.

The presented protocols can be used to characterize the response of fluorescently-labeled microbubbles designed for ultrasound-triggered drug delivery applications, including their activation mechanisms as well as their bioeffects. This paper covers a range of in vitro and in vivo microscopy techniques performed to capture the relevant length and timescales.

Microbubble contrast agents hold great promise for drug delivery applications with ultrasound. Encapsulating drugs in nanoparticles reduces systemic ...