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

Abstract

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 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 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.

Characterization of Anti-Angiogenic Chemo-Sensitization via Longitudinal Ultrasound Localization Microscopy in Colorectal Carcinoma Tumor Xenografts

“…The chicken embryo was then immediately moved to the adjacent ultrasound imaging system for ULM acquisition, where the transducer was placed to produce the largest imaging cross-section of the tumor. It has been demonstrated that microbubbles will remain in circulation for several hours in the chicken embryo [ 34 ], so although the time delay between the microscope-guided microbubble injection and ultrasound imaging precludes conventional contrast-ultrasound indicator-dilution quantifications, there will remain sufficient recirculating microbubbles for ULM reconstruction. Each CAM tumor was imaged using 20 separate acquisitions of 1600 frames each, resulting in a total acquisition length of 32000 frames (32 seconds) per imaging session.…”

“…Although there was some chicken embryo attrition noted in all of the randomized treatment groups (Figure 1C), this study still had 34 HCT-116 tumors and 29 HT-29 tumors for the endpoint (post-treatment) imaging session on EDD-17, with at least N ≥ 5 in each treatment group. This level of attrition is not unexpected for the ex ovo chicken embryo model, which routinely sees attrition rates approaching 50% [49], but the low relative cost of chicken embryo xenograft models allows for a large safety margin in study planning to ensure that there will be sufficient samples at the endpoint.…”

3-D contrast enhanced ultrasound imaging of an in vivo chicken embryo with a sparse array and deep learning based adaptive beamforming

“…An ex ovo chicken embryo and chorioallantoic membrane (CAM) were used as the in vivo model to investigate the effect of the different beamformers on contrast enhanced ultrasound imaging. The five-day-old chicken embryo and CAM were removed from the eggshell and further prepared according to the protocol of [8], after which four µL of custom-made F-type microbubble was injected into the vasculature of the chicken embryo. The chicken embryo was placed in a PBS solution and the ultrasound array was positioned on top of the chicken embryo at a 3 cm distance.…”

Theranostic Microbubbles with Homogeneous Ligand Distribution for Higher Binding Efficacy

“…For in vivo experiments, the CAM was prepared as described previously [42]. All animal experiments were conducted in accordance with The Netherlands Experiments on Animals Act and in accordance with the European Council (2010/63/EU) on the protection of animal use for scientific purposes.…”