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

Civciv Embriyo Beynini İnsan Glioblastoma Hücre Davranışını İncelemek İçin Bir Model Olarak Kullanma

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.

Yazar Spotlight: Civciv Embriyo Beyninin İnsan Glioblastoma Hücre Davranışının İn Vivo ve Ex Vivo Analizleri için Bir Model Olarak Kullanılması  

Civciv Embriyo Beynini, İnsan Glioblastoma Hücre Davranışının In Vivo ve Ex Vivo Analizleri için Bir Model Olarak Kullanma

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  

Ovo'da E5 Optik Tektuma Glioblastoma (GBM) Hücrelerinin Enjeksiyonu

Başlamak için, döllenmiş tavuk yumurtalarını nemlendirilmiş bir yumurta inkübatöründe, sivri ucu 37.5 santigrat dereceye bakacak şekilde inkübe edin. Kuluçkanın altıncı gününde veya E5, yumurta kabuğunu% 70 etanol ile püskürterek sterilize edin. Bir yumurta mumcusu kullanarak, embriyonun üzerindeki hava sahasının çevresi boyunca bir kalemle iz bırakın ve ana hatlarıyla belirtilen alanı şeffaf bantla örtün.Embriyonik zarları veya kan damarlarını kesmeden izlenen alanın etrafını kavisli makasla yavaşça kesin. Yumurta kabuğunun üstünü atın. Hava sahası zarına birkaç damla salin veya hücre kültürü ortamı ekleyerek kolayca ayrılabilmesi için ıslatın.İnce forseps kullanarak, hava sahası zarını embriyonun tepesinden dikkatlice delin. Kaldırın. Kafayı konumlandırmak için embriyoyu hemen çevreleyen şeffaf amniyon zarını alın ve beş mikrolitre uygun hücre kültürü ortamında yaklaşık 50.000 hücreyi bir cam mikropipet ve bir Pnömatik PicoPump ile optik tektuma enjekte edin.Embriyonun üzerine mililitre ampisilin başına birkaç damla 50 miligram ekleyin ve yumurtaların üstündeki deliği şeffaf bantla örtün. Diseksiyon için E15'e kadar nemlendirilmiş bir yumurta inkübatörüne yerleştirin. Glioblastoma veya GBM kök hücrelerinin enjeksiyonundan sonra E5 optik tektumunda in vivo olarak oluşan tümörler veya E15'te yeşil floresan proteini eksprese eden GSC'ler bu şekilde gösterilmiştir.GSC'ler ventriküler yüzeye yapışır ve beyin duvarında invaziv tümörler oluşturur. Bu deneydeki beş özelliği tanımlamak için dört renk kullanıldı: yeşil GSC'ler, beyaz çekirdekler, beyaz kan damarları, mavi integrin alfa-6 ve kırmızı SOX2 veya kırmızı Nestin. Bu rakamlar, GSC'lerin kan damarlarınızda bulunduğunu ve bunlar boyunca göç ediyor gibi göründüğünü göstermektedir.İn vivo GSC tümörlerinin sabit ve immün boyalı dilimlerine dönen 3D hacim render filmleri burada gösterilmektedir.

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

538 Görüntüleme. Başlamak için, döllenmiş tavuk yumurtalarını nemlendirilmiş bir yumurta inkübatöründe, sivri ucu 37.5 santigrat dereceye bakacak şekilde inkübe edin. Kuluçkanın altıncı gününde veya E5, yumurta kabuğunu% 70 etanol ile püskürterek sterilize edin. Bir yumurta mumcusu kullanarak, embriyonun üzerindeki hava sahasının çevresi boyunca bir kalemle iz bırakın ve ana hatlarıyla belirtilen alanı şeffaf bantla örtün.Embriyonik zarları veya kan damarlarını kesmed...

Video: 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 ...