Name: Renal Organotypic Culture on Chicken CAM: A Technique for Transplantation of Murine Kidney Explants on Chicken CAM to Induce Formation of Renal Vascular Architecture
Uploaded: 2023-04-30T13:54:35
Description: Source: Kaisto, S. et al. Optimization of Renal Organoid and Organotypic Culture for Vascularization, Extended Development, and Improved Microscopy ...

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board
1. Fabrication of Minireservoirs for Cultivation of Mouse Embryonic Kidneys and Renal Organoids on Chicken CAM and Setting Up Xenotransplantation Experiments
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	<li>Use transwell cell culture inserts designed for 6-well or 12-well plates. 
	NOTE: Depending on the particular experiment, large or small minirese...

renal organotypic culture on chicken cam: a technique for transplantation of murine kidney explants on chicken cam to induce formation of renal vascular architecture

Source: Kaisto, S. et al. <a href="https://www.jove.com/video/60995" target="_blank">Optimization of Renal Organoid and Organotypic Culture for Vascularization, Extended Development, and Improved Microscopy Imaging.</a> J. Vis. Exp. (2020)
This video describes a method for studying organ development using an improved xenotransplantation setup on chorioallantoic membrane (CAM) from avian embryos. This method allows for the development of fully vascularized cultured embryonic organs and organoids.

Renal Organotypic Culture on Chicken CAM: A Technique for Transplantation of Murine Kidney Explants on Chicken CAM to Induce Formation of Renal Vascular Architecture

Source: Kaisto, S. et al. Optimization of Renal Organoid and Organotypic Culture for Vascularization, Extended Development, and Improved Microscopy ...

Chicken chorioallantoic membrane, or CAM, is a highly vascularized and naturally immunodeficient layer present beneath the eggshell. CAM serves as an ideal model for xenografting - a heterologous transfer of tissue from a donor organism to the recipient of another species.
To perform renal organotypic culture on chicken CAM, begin by taking a mini-reservoir with a permeable membrane present at the base. Invert the mini-reservoir and position it over a drop of media present in a Petri dish. This ensures that the membrane is in direct contact with the media. Transfer the murine kidney explants to the pre-wet membrane. Aspirate the excess media around the kidneys.
Incubate to facilitate the attachment of the kidney explants to the membrane. Next, take an ex-ovo culture from an embryonic chicken exhibiting a fully developed CAM. Locate the prominent blood vessels peripheral to the embryo. Take the reservoir with attached kidney explants and invert it over the CAM such that the membrane presses the kidney explants against the CAM tissue.
Supplement the reservoir with media to support the growth of kidney explants and prevent their shrinkage. Cultivate for a prolonged duration. The CAM blood vessels infiltrate the transplanted kidney and induce vascularization within the kidney explants, stimulating the functional kidney&#39;s recapitulation on CAM tissue.

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Ex vivo studies

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

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Neuroscience

Comparing Metastatic Clear Cell Renal Cell Carcinoma Model Established in Mouse Kidney and on Chicken Chorioallantoic Membrane

Metastatic clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Localized ccRCC has a favorable surgical outcome. However, one third of ccRCC patients will develop metastases to the lung, which is related to a very poor outcome for patients. Unfortunately, no therapy is available for this deadly stage, because the molecular mechanism of metastasis remains unknown. It has been known for 25 years that the loss of function of the von Hippel-Lindau (VHL) tumor suppressor gene is pathognomonic of ccRCC. However, no clinically relevant transgenic mouse model of ccRCC has been generated. The purpose of this protocol is to introduce and compare two newly established animal models for metastatic ccRCC. The first is renal implantation in the mouse model. In our laboratory, the CRISPR gene editing system was utilized to knock out the VHL gene in several RCC cell lines. Orthotopic implantation of heterogeneous ccRCC populations to the renal capsule created novel ccRCC models that develop robust lung metastases in immunocompetent mice. The second model is the chicken chorioallantoic membrane (CAM) system. In comparison to the mouse model, this model is more time, labor, and cost-efficient. This model also supported robust tumor formation and intravasation. Due to the short 10 day period of tumor growth in CAM, no overt metastasis was observed by immunohistochemistry (IHC) in the collected embryo tissues. However, when tumor growth was extended by two weeks in the hatched chicken, micrometastatic ccRCC lesions were observed by IHC in the lungs. These two novel preclinical models will be useful to further study the molecular mechanism behind metastasis, as well as to establish new, patient-derived xenografts (PDXs) toward the development of novel treatments for metastatic ccRCC.

Metastatic clear cell renal cell carcinoma is a disease without a comprehensive animal model for thorough preclinical investigation. This protocol illustrates two novel animal models for the disease: the orthotopically implanted mouse model and the chicken chorioallantoic membrane model, both of which demonstrate lung metastasis resembling clinical cases.

Metastatic clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Localized ccRCC has a favorable surgical outcome. However, ...

Using the Chicken Chorioallantoic Membrane In Vivo Model to Study Gynecological and Urological Cancers

Mouse models are the benchmark tests for in vivo cancer studies. However, cost, time, and ethical considerations have led to calls for alternative in vivo cancer models. The chicken chorioallantoic membrane (CAM) model provides an inexpensive, rapid alternative that permits direct visualization of tumor development and is suitable for in vivo imaging. As such, we sought to develop an optimized protocol for engrafting gynecological and urological tumors into this model, which we present here. Approximately 7 days postfertilization, the air cell is moved to the vascularized side of the egg, where an opening is created in the shell. Tumors from murine and human cell lines and primary tissues can then be engrafted. These are typically seeded in a mixture of extracellular matrix and medium to avoid cellular dispersal and provide nutrient support until the cells recruit a vascular supply. Tumors may then grow for up to an additional 14 days prior to the eggs hatching. By implanting cells stably transduced with firefly luciferase, bioluminescence imaging can be used for the sensitive detection of tumor growth on the membrane and cancer cell spread throughout the embryo. This model can potentially be used to study tumorigenicity, invasion, metastasis, and therapeutic effectiveness. The chicken CAM model requires significantly less time and financial resources compared to traditional murine models. Because the eggs are immunocompromised and immune tolerant, tissues from any organism can potentially be implanted without costly transgenic animals (e.g., mice) required for implantation of human tissues. However, many of the advantages of this model could potentially also be limitations, including the short tumor generation time and immunocompromised/immune tolerant status. Additionally, although all tumor types presented here engraft in the chicken chorioallantoic membrane model, they do so with varying degrees of tumor growth.

We present the chicken chorioallantoic membrane model as an alternative, transplantable, in vivo model for the engraftment of gynecological and urological cancer cell lines and patient-derived tumors.

Mouse models are the benchmark tests for in vivo cancer studies. However, cost, time, and ethical considerations have led to calls for alternative in vivo ...

Renal Organotypic Culture on Chicken CAM: A Technique for Transplantation of Murine Kidney Explants on Chicken CAM to Induce Formation of Renal Vascular Architecture

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