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

ヒト膠芽腫細胞の挙動を研究するためのモデルとしてニワトリ胚脳を使用

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.

著者スポットライト:ヒト膠芽腫細胞の挙動のIn vivoおよびex vivo解析のためのモデルとしてのニワトリ胚脳の使用  

ニワトリ胚脳をモデルとしてヒト膠芽腫細胞挙動の in vivoおよび ex vivo 解析(英語)

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  

膠芽腫(GBM)細胞のOvoのE5視神経胸部への注射

まず、尖った端を摂氏37.5度で下に向けて加湿卵インキュベーターで受精鶏卵を孵化させます。孵卵の6日目、またはE5に、70%エタノールを噴霧して卵殻を殺菌します。卵キャンドラーを使用して、胚の上の空域の周囲に沿って鉛筆でトレースし、輪郭を描かれた領域を透明なテープで覆います。胚膜や血管に切り込まずに、湾曲したハサミでトレース領域の周りをそっと切ります。卵殻の上部を捨てます。生理食塩水または細胞培養培地を空域膜に数滴加えて濡らし、簡単に剥離できるようにします。細かい鉗子を使用して、胚の上に空域膜を慎重に突き刺します。それを削除します。胚をすぐに囲む透明な羊膜をつかんで頭を配置し、ガラスマイクロピペットと空気圧PicoPumpを使用して、5マイクロリットルの適切な細胞培養培地で約50, 000個の細胞を視蓋に注入します。胚の上にアンピシリン1ミリリットルあたり50ミリグラムを数滴加え、卵の上の穴を透明なテープで覆います。解剖のためにE15まで加湿卵インキュベーターにそれらを置きます。膠芽腫、GBM幹細胞、またはE15で緑色蛍光タンパク質を発現するGSCの注射後にin vivoでE5視蓋に形成された腫瘍をこの図に示します。GSCは心室表面に付着し、脳壁に浸潤性腫瘍を形成します。この実験では、緑色のGSC、白い核、白い血管、青いインテグリンα-6、および赤色のSOX2または赤色のネスチンの4つの特徴を識別するために使用されました。これらの図は、GSCが血管内に存在し、血管に沿って移動しているように見えることを示しています。ここでは、in vivo GSC腫瘍の固定および免疫染色されたスライスに回転する3Dボリュームレンダリングの動画を示します。

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

550 ビュー. まず、尖った端を摂氏37.5度で下に向けて加湿卵インキュベーターで受精鶏卵を孵化させます。孵卵の6日目、またはE5に、70%エタノールを噴霧して卵殻を殺菌します。卵キャンドラーを使用して、胚の上の空域の周囲に沿って鉛筆でトレースし、輪郭を描かれた領域を透明なテープで覆います。胚膜や血管に切り込まずに、湾曲したハサミでトレース領域の周りをそ...

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

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

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 ...