injection of glioblastoma (gbm) cells into e5 optic tectum in ovo

Using Chick Embryo Brain as a Model to Study Human Glioblastoma Cell Behavior

In vitro studies of cancer cell behaviors are often used to dissect potential mechanisms that operate during the more complex behavior that is observed during tumor formation and cell invasion in in vivo xenograft models. For example, with glioblastoma (GBM), in vitro studies have uncovered mechanisms of how L1CAM potentially operates during tumor formation and brain invasion in a novel chick embryo xenograft brain tumor model1,2,3,4,5. Although in vitro and in vivo experiments complement each other in useful ways, they leave a substantial gap in how the results can be correlated. For instance, mechanistic analyses of GBM cell motility on a dish is a highly artificial situation, and in vivo xenograft models can only reveal static time point or endpoint analyses of tumor formation and cell behavior. In vivo studies using either rodents or chick embryos do not easily lend themselves to monitoring cell behavior while the cells invade brain tissue in these xenograft models. Nevertheless, the chick embryo xenograft model has demonstrated that the adhesion protein L1CAM plays a stimulatory role in the invasive ability of human T98G GBM cells2,5.
A suitable solution to this problem can be reached by bridging both in vivo and in vitro methods using an organotypic brain slice culture model, referred to as an ex vivo model. In this ex vivo model, live brain tissue can be maintained at a thickness of several hundred microns for up to a few weeks, making it possible to implant cancer cells, observe their behavior in actual tissue over time, and then perform a more detailed marker analysis at the endpoint of the experiment.
A popular organotypic slice culture method has been to culture a several hundred micron-thick brain slice on top of a translucent or transparent porous membrane, leaving the tissue exposed to air, yet allowing nutrient media to sustain the tissue from below the membrane (refer to Stoppini et al.6). Different variations of this method have been used for different studies, including using different media or different membrane inserts. Different membrane inserts include a 30 mm diameter, porous (0.4 &#181;m) membrane insert in a 35 mm culture dish6, and cell culture inserts (0.4 &#181;m) for 6-well plates7. Different media include 50% MEM/HEPES + 25% heat-inactivated horse serum + 25% Hanks balanced salt solution (HBSS)8, 50% reduced serum media + 25% horse serum + 25% HBSS9, as well as others. If a translucent or transparent membrane is used along with fluorescently labeled GBM cells, then such cultures can be imaged from below using an inverted widefield or confocal fluorescence microscope10,11,12,13,14,15.
While many in vivo orthotopic brain tumor xenograft and ex vivo organotypic brain slice culture models have been established using rodents, as cited above, the chick embryo (Gallus gallus) has been underutilized for these purposes. However, the chick embryo has been demonstrated to be capable of being used as an in vivo orthotopic xenograft model for the study of both human and rat glioma invasion1,2,5. Xenografted cells into chick embryo brains have exhibited invasion patterns similar to those observed in rodent models, further supporting the use of chick embryos as an in vivo model for GBM tumor cell analysis. Chick embryos also are inexpensive, can be more easily maintained than rodents (i.e., in their egg shells in a lab incubator), and are much easier to work with, making them an attractive option for short-term in vivo GBM studies. A recent article has described the use of chick embryo brain slice cultures for the formation and growth of axons during normal brain development where the slices were viable for at least 7 days16. However, the use of such chick embryo brain slice cultures for ex vivo analysis of GBM cell behavior in a tissue environment is lacking. In this article, both the transplantation of human GBM cells and GBM stem cells (GSCs) into the early chick embryo brain in vivo, as well as the introduction of GBM cells onto live chick embryo brain slice cultures ex vivo, are described. Some representative examples of the resulting tumors and cell invasion patterns obtained from these preparations are also provided.

Author Spotlight: Using the Chick Embryo Brain as a Model for In Vivo and Ex Vivo Analyses of Human Glioblastoma Cell Behavior  

Using the Chick Embryo Brain as a Model for In Vivo and Ex Vivo Analyses of Human Glioblastoma Cell Behavior

The scope of our research is to try to uncover mechanisms that promote glioblastoma cell spread in brain tissue. Specific questions revolve around differences in glioblastoma cell types or states and molecular mechanisms that promote these cells to be highly invasive. For example, how does L1CAM expression promote invasiveness?Both in vitro and in vivo models are insufficient for accurately analyzing glioblastoma cell behavior. In vitro models almost certainly modify and oversimplify in vivo cell behaviors. While in vivo models do not allow for easy observation of behaviors as they occur, instead allowing for analyses after the behaviors have occurred.We have shown that the chick embryo brain is a good model for studying human brain cancer cell behavior both in vivo and in ex-vivo slice cultures. We also have established that L1CAM expression by glioblastoma cells can have profound effects on cell proliferation, invasion, and arrangement within the tumor. Besides several advantages of using chick embryos, our ex-vivo spheroid protocol introduces cells onto live slices with no additional damage, whereas other methods of cell introduction involve piercing the tissue and implanting cells.Additionally, the protocol allows for live time-lapse imaging that in vivo techniques do not. Our findings suggests that the chick embryo is a good model for cancer research, particularly brain cancer, and is extremely beneficial for the scientific community, as it is much more readily available to those who may not have the funds, facilities, or expertise for rodent models. We will continue to focus on molecular mechanisms that control glioblastoma cell invasiveness in brain tissue, particularly along blood vessels.Another crucial question is whether glioblastoma stem cells are a distinct and stable phenotype or a functional state that can be adopted when needed. That has huge implications for treatments.

Watch this Scientific Journal Video about Injection of Glioblastoma GBM Cells into E5 Optic Tectum In Ovo at JoVE.com

Injection of Glioblastoma (GBM) Cells into E5 Optic Tectum In Ovo  

To begin, incubate the fertilized chicken eggs in a humidified egg incubator with the pointed end facing down at 37.5 degrees Celsius. On the sixth day of incubation, or E5, sterilize the eggshell by spraying it with 70%ethanol. Using an egg candler, trace along the perimeter of the airspace above the embryo with a pencil and cover the outlined area with transparent tape.Gently cut around the traced area with curved scissors without cutting into the embryonic membranes or blood vessels. Discard the top of the eggshell. Add a few drops of saline or cell culture media onto the airspace membrane to wet it so that it will detach easily.Using fine forceps, carefully pierce the airspace membrane over the top of the embryo. Remove it. Grab the transparent amnion membrane that immediately surrounds the embryo to position the head and inject around 50, 000 cells in five microliters of suitable cell culture media into the optic tectum with a glass micropipette and a Pneumatic PicoPump.Add a few drops of 50 milligrams per milliliter of ampicillin on top of the embryo and cover the hole in the top of the eggs with clear tape. Place them in a humidified egg incubator until E15 for dissection. The tumors that formed in the E5 optic tectum in vivo after the injection of glioblastoma, or GBM stem cells, or GSCs expressing green fluorescent protein at E15 are shown in this figure.GSCs attach to the ventricular surface and form invasive tumors in the brain wall. Four colors were used to identify five features in this experiment, green GSCs, white nuclei, white blood vessels, blue integrin alpha-6, and either red SOX2 or red Nestin. These figures show that the GSCs reside in your blood vessels and appear to be migrating along them.Movies of rotating 3D volume renders to fixed and immunostained slices of in vivo GSC tumors are shown here.

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550 Views.  University of Delaware. To begin, incubate the fertilized chicken eggs in a humidified egg incubator with the pointed end facing down at 37.5 degrees Celsius. On the sixth day of incubation, or E5, sterilize the eggshell by spraying it with 70%ethanol. Using an egg candler, trace along the perimeter of the airspace above the embryo with a pencil and cover the outlined area with transparent tape.Gently cut around the traced area with curved scissors without cutting into the embryonic membranes or blood vessels...

Video: Injection of Glioblastoma (GBM) Cells into E5 Optic Tectum In Ovo  

The chick embryo has been an ideal model system for the study of vertebrate development, particularly for experimental manipulations. Use of the chick embryo has been extended for studying the formation of human glioblastoma (GBM) brain tumors in vivo and the invasiveness of tumor cells into surrounding brain tissue. GBM tumors can be formed by injection of a suspension of fluorescently labeled cells into the E5 midbrain (optic tectum) ventricle in ovo. 
Depending on the GBM cells, compact tumors randomly form in the ventricle and within the brain wall, and groups of cells invade the brain wall tissue. Thick tissue sections (350 &#181;m) of fixed E15 tecta with tumors can be immunostained to reveal that invading cells often migrate along blood vessels when analyzed by 3D reconstruction of confocal z-stack images. Live E15 midbrain and forebrain slices (250-350 &#181;m) can be cultured on membrane inserts, where fluorescently labeled GBM cells can be introduced into non-random locations to provide ex vivo co-cultures to analyze cell invasion, which also can occur along blood vessels, over a period of about 1 week. These ex vivo co-cultures can be monitored by widefield or confocal fluorescence time-lapse microscopy to observe live cell behavior. 
Co-cultured slices then can be fixed, immunostained, and analyzed by confocal microscopy to determine whether or not the invasion occurred along blood vessels or axons. Additionally, the co-culture system can be used for investigating potential cell-cell interactions by placing aggregates of different cell types and colors in different precise locations and observing cell movements. Drug treatments can be performed on ex vivo cultures, whereas these treatments are not compatible with the in ovo system. These two complementary approaches allow for detailed and precise analyses of human GBM cell behavior and tumor formation in a highly manipulatable vertebrate brain environment.

Chick embryos are used for studying human glioblastoma (GBM) brain tumors in ovo and in ex vivo brain slice co-cultures. GBM cell behavior can be recorded by time-lapse microscopy in ex vivo co-cultures, and both preparations can be analyzed at the experimental endpoint by detailed 3D confocal analysis.

The chick embryo has been an ideal model system for the study of vertebrate development, particularly for experimental manipulations. Use of the chick ...

Live Chick Embryo Brain Dissection and Vibratome Slicing

Use an egg candler to trace along the perimeter of the air pocket above the E14 or E15 embryo with a pencil and cover the outlined area with transparent tape. Using curved or fine scissors, gently cut around the traced area. Be careful not to cut into the embryo membrane or blood vessels.Then discard the top piece of the shell. After decapitating the chick embryo, place the head in a 10 centimeter dish with a cold, sterile CMF solution. Using sterile fine forceps, peel away the skin covering the brain to reveal the underlying dura mater.Remove the forming skull bones on the left and right sides of the brain. The forming skull bones do not yet cover the majority of the brain. Gently use fine pointed forceps to tear through the meninges, overlying the center of the brain and peel it to each side to uncover the brain.Using fine curved forceps, scoop the brain up from the bottom front and gently pull it out of its cavity. Dissect the brain into its three main parts forebrain, midbrain, or optic tecta and cerebellum. Remove the overlying pia mater from the tecta using fine forceps and place the dissected brain regions in a small sterile dish on ice.For embedding and slicing the brain, gently pick up either the optic tectum or forebrain region with curved forceps as a scoop and blot slightly on sterile gauze to remove extra liquid. Prepare a simple mold by forming aluminum foil around an appropriately sized object. Here, a small rectangular metal vibrating tissue slicer block is used as the object.Use a sterile transfer pipette and fill the mold with low melt agarose. Quickly place the brain region in agarose and let it solidify for approximately four to five minutes on ice. Cut the sections on a vibrating tissue slicer using a sapphire knife.Use the sterile spatula to scoop up and remove the slice from the tray. Gently slide the section off the spatula and onto a membrane insert using another sterile spatula. Place the six well plate of brain slices on membrane inserts in the cell culture incubator at 37 degrees Celsius and 5%carbon dioxide.On the day after platting, use a sterile Pasteur pipette to aspirate the media from below the insert and add one milliliter of fresh slice media to each well underneath the membrane insert. Next, to introduce the GBM cells onto the brain slices, remove one to several spheroids from their culture dish using a 20 microliter micro pipette set to five microliters. Gently expel the media with spheroids onto the desired brain slice and allow the spheroids to culture on the slice for two to five days.The viability results of x vivo optic tectum slices after one week in culture are shown in this figure. The confocal images of a brain slice stained for nuclei with bisbenzimide and immunostained for laminin clearly shows normal and intact blood vessels optically sectioned in various configurations. The maximum projection image of the confocal Z stack of the brain slice stained for SOX 2 transcription factor and total nuclei with bisbenzimide is shown here.These results suggest that chick embryo brain slices could be cultured on membrane inserts for approximately two weeks and remain viable with normal appearing blood vessels and transcription factor expression.