Cells from the SW1353 chondrosarcoma cell line were kindly provided by Prof. Dr. P.C.W. Hogendoorn and Prof. Dr. J. Bov&eacute;e of Leiden University, The Netherlands. We thank J. Mestach and G. Wagemans for excellent technical assistance, and G. De Bruyne for the professional drawing of the overview of our protocol.

See Figure 1 for an overview.
Tumor material
1. Obtaining and Preparing Tumor Samples
For the use of patient material, approval of the Ethical Committee is necessary, and informed consent has to be obtained from the patient.
<ol>
 <li>Harvest representative material (minimum 1 cm3) at the time of intervention, either a biopsy or a resection of a sarcoma. The proper site of biopsy is defined using dynamic-contrast MRI.</li>
 <li>Place the material immediately in a sterile vial containing DMEM supplemented with 1,000 U Penicillin and 1 mg/ml streptomycin.</li>
 <li>Transport the vial immediately (30 - 90 min) to the lab.</li>
</ol>
All following procedures are performed under laminar flow.
<ol start="4">
 <li>Pour the contents of the vial into a sterile Petri dish.</li>
 <li>Put the tumor sample into small parts of maximally 1 mm3 using a sterile scalpel. Remove all calcified parts as they tend to impair further processing of the tissue.</li>
 <li>Randomly take 10 tumor grafts for application on the CAM.</li>
</ol>
2. Preparation of Tumor-derived Single Cell Suspensions
Approximate duration: 3 hr.
<ol>
 <li>Weigh the remaining tissue of the sample.</li>
 <li>Put 2 - 4 g of tissue in a dissociator tube, more than one tube may be required to digest all the remaining tumor tissue.</li>
 <li>For digestion of 2 - 4 g of tissue, add 2.5 ml collagenase 2 solution (500 U/ml RPMI 1640) and 2.5 ml DNase solution (22 KU/ml RPMI 1640).</li>
 <li>Process the tissue according to the dissociator h_tumor protocol. This involves 2 cycles of incubation at 37 &deg;C in CO2 incubator while the tube is shaken gently for 30 min.</li>
 <li>Filter the remaining suspension through a 150 &mu;m cell strainer.</li>
 <li>Centrifuge the lysate at 1,000 rpm for 5 min.</li>
 <li>Remove the supernatant.</li>
 <li>Resuspend the pellet in 10 ml of erythrocyte lysis buffer (ELB), allowing 10 min of incubation with regular suspension.</li>
</ol>
Note: erythrocyte lysis buffer is manufactured as follows: Add 0.037 g EDTA, 0.99 g K2HPO4, 8.29 g NH4Cl to 1,000 ml aqua, use 10 N NaOH to adjust the pH to 7.3, filter under pressure through a 0.22 &mu;m filter. Store at 4 &deg;C.
<ol start="9">
 <li>Add 25 ml of culture medium (DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 &mu;g/ml streptomycin) to stop the reaction.</li>
 <li>Centrifuge the suspension at 1,000 rpm for 5 min. The color of the pellet should be white now.</li>
 <li>Remove the supernatant.</li>
 <li>Add 5 ml of medium to the pellet. Filter the suspension through a 70 &mu;m cell strainer.</li>
 <li>Count the number of live cells/ml by means of an automatic cell counter.</li>
</ol>
3. Cancer Cell Lines
SAOS2 osteosarcoma cells (ATCC number: HTB-85) expressing enhanced green fluorescent protein were electroporated by peGFP-C1 vector using the Cell Line Nucleofector Kit V according to the manufacturer's protocol. To establish stable cell lines, transfected cells were selected in G418 (1 mg/ml) for 4 weeks in line with previous established protocol20. In addition sorting was performed by fluorescence activated cell sorting (FACS) according to the manufacturer's instructions.
Cells from the SW1353 chondrosarcoma cell line (ATCC number: HTB-94) or freshly prepared tumor single cell suspensions are labeled using a red fluorescent lipophilic membrane stain.
<ol>
 <li>Remove the cells from the culture flask in a classical way using Trypsin (0.5% wt/vol) ethylenediaminetetraacetic acid (EDTA; 0.2% wt/vol) solution.</li>
 <li>Centrifuge the cell suspension for 5 min at 1,000 rpm.</li>
 <li>Remove the supernatant.</li>
 <li>Add 1 ml of serum-free medium and 5 &mu;l DiI (Vybrant Red)/106 cells.</li>
 <li>Wrap the vial in aluminum foil and put it in the CO2 incubator for 10 min at 37 &deg;C.</li>
 <li>Centrifuge again for 5 min at 1,000 rpm and remove the supernatant.</li>
</ol>
Chorioallantoic membrane assay
1. Egg Incubation
<ol>
 <li>Fertilized eggs from a local commercial hatchery guaranteeing 95% fertilisation of the eggs are required.</li>
 <li>Fertilized eggs can be stored at RT up to 10 days.</li>
</ol>
Note: before starting incubation, dirt, feathers and excrement are carefully removed from the eggshells mechanically by dry wiping with paper towels, which have a rough rather than a soft surface structure. Wiping the eggs with 70% denatured ethanol or any other cleaning reagent significantly reduces the survival rate of the chick embryos.
<ol start="3">
 <li>Put the eggs in the incubator on one side, marking the upper surface. The day on which the eggs are put in the incubator is designated day 0 of embryonic development.</li>
 <li>Set the incubation temperature at 37.8 &deg;C, and the humidity at 47%. Make sure the automatic rotation option is switched off.</li>
</ol>
2. Opening of the Eggs
Duration: &plusmn; 60 min for 25 eggs.
On development day 3, the eggs are opened under laminar airflow. Opening the eggs at a further developmental stage results in damage to the CAM, as the membrane tends to stick to the shell. An infrared lamp is used to keep the eggs warm during the procedure. To improve sterility, we recommend the use of hand gloves.
<ol>
 <li>Place the egg in the eggcup, with the marked side facing upwards.</li>
 <li>Lower the level of the CAM by removing two ml of albumen through an 18 G needle inserted at the tip of the egg. If the needle is blunt after perforating several eggshells, replace the needle. If not, too much force has to be exerted, resulting in damage to the egg yolk or fracture of the shell. Sometimes the needle is blocked by the chalazae, one of 2 spiral bands suspending the yolk in albumen. This can be solved by gently pushing the plunger back down until the block is resolved. If egg yolk is sucked into the syringe, replace the needle as well as the syringe. Sometimes the embryo dies after this event. If an insufficient amount of albumen is removed, the CAM will be damaged when the shell is removed.</li>
 <li>Apply a semipermeable adhesive film at the marked upper side of the shell in order to prevent spilling of shell particles onto the CAM while cutting the window.</li>
 <li>Make an introduction hole with a small awl at the surface of the egg.</li>
 <li>Cut a window of 1 cm2 in the shell, using a pair of sterile sharp-pointed surgical scissors.</li>
 <li>The embryo's pulsating heart and adjoining vessels can be observed at the surface of the egg yolk. Remove the non-fertilized eggs or dead embryos. An interrupted aspect of the blood vessels characterizes dead embryos.</li>
 <li>Seal the window with a semipermeable adhesive film. It is not necessary to cover the puncture site, unless the shell has cracked during insertion of the needle.</li>
</ol>
3. Inoculation Procedure
Duration: &plusmn; 1 hr per cell line.
On chick embryonic development day 9, the eggs are inoculated under laminar airflow.
Evaluation of cancer cell spreading across the CAM requires containment of the cells to a limited surface during inoculation. Matrigel is an extracellular matrix (ECM) gel from the Engelbreth-Holm-Swarm mouse sarcoma frequently used in experimental cell culture conditions. It is a fluid when cooled, but will undergo thermally activated polymerization when brought to 20 - 40 &deg;C, thus forming a stable gel. During the experiments, Matrigel is kept in a box containing melting ice.
Note: We insist not to use perforated coverslips. We observed attachment and growth of grafted cancer cells onto the plastic disc thus biasing growth potential of the cells on the membrane itself.
<ol>
 <li>Resuspend a cell pellet in cooled ECM gel at a concentration of 106 cells/100 &mu;l ECM gel. Keep this solution on melting ice.</li>
 <li>Excise part of the semi-permeable membrane with a pair of sterile surgical under laminar airflow. If the adhesive film is removed completely, this will result in a larger proportion of embryonic death due to contamination.</li>
 <li>Touch the CAM right below the shell window lightly with a sterile glass rod, in order to remove the epithelial cell layer. Touching the CAM with a sterile glass rod proves to be less traumatic than using a scalpel n&deg; 11, which requires more handiness and experience. Small hemorrhages may occur.</li>
 <li>Adding tumor material.</li>
 <li>For tumor grafts: gently lower one piece of tissue onto the CAM with a pair of sterile forceps, without squeezing it.</li>
 <li>For the ECM gel procedure: Add 4x 25 &mu;l of the cell-ECM gel suspension to the CAM, on top of each other, allowing the ECM gel to polymerize in between the applications. In this way an adherent plaque containing the cancer cells is created.</li>
 <li>Seal the shell window again with a semipermeable adhesive film.</li>
</ol>
4. Imaging and Harvesting of the CAM
Duration: 2 hr for 25 eggs.
On chick embryonic development day 16, the CAMs are harvested. Working under laminar airflow is not necessary.
<ol>
 <li>Enlarge the window with a pair of surgical scissors. Make sure not to cut too low, otherwise the CAM will be damaged, resulting in bleeding of the injured vessels.</li>
 <li>Add 300 - 500 &mu;l of PBSD with a pipette onto the CAM section containing the inoculated material. If no fluorescent probes are used, buffered formaldehyde may be used as this makes the membrane stiffer, facilitating the cutting of the membrane.</li>
 <li>Make a photograph of the CAM in the egg through a stereo fluorescence microscope, equipped with a digital color camera, using both the GFP or the DSR filter, and no filter. For the tumor grafts, all photographs are made without filters. Illumination from above by LED lights is strongly recommended during photographing (Figure 2).</li>
 <li>Record tumor cell migration for the labeled cells. The borders of the tumors may be irregular and frayed. Scattered cells may be present near the tumor borders. In some specimens, rows of tumor cells may be observed (Figure 3).</li>
 <li>Cut the membrane with a pair of sterile scissors at the border lying against the shell.</li>
 <li>Place the harvested CAM in a sterile Petri dish filled with PBSD or buffered formaldehyde.</li>
 <li>Make a photograph of both the upper side and the lower side of the CAM, both with and without filter.</li>
 <li>Put the membrane in a labeled container with buffered formaldehyde for at least 72 hr.</li>
 <li>Embed the CAMs in paraffin and prepare them for histological examination. Take care to cut the tumor grafts at the center of the nodule, making sure the cut surface is parallel to the bottom of the cassette. If not, the interaction between the CAM and the tumor graft will not be visible. The same strategy applies to the ECM gel plaques: the surface facing the cutting blade should be perpendicular to the plaque.</li>
</ol>
5. Histological Evaluation
Hematoxylin-Eosin staining and Ki-67 immunohistochemistry were performed for further clarification, if needed. Immunohistochemistry was performed on formalin-fixed paraffin-embedded tissue sections of 3.5 &mu;m in thickness, using an automated slide stainer according to manufacturer's instructions. A mouse primary monoclonal antibody to Ki-67 (clone MIB-1, dilution 1/100) was used. Heat-induced epitope retrieval was performed using Cell Conditioning 1, and visualization was achieved with the Universal DAB Detection Kit, according to manufacturer's instructions. Dehydratation of the tissue sections was carried out using an automated coverslipper.
For the SAOS2 and the SW1353 cell lines, the number of Ki67-positive and Ki67-negative cell nuclei/100 &mu;m2 were counted in three zones: one close to the border of the CAM, one close the surface and one in the center of the ECM gel plaque. This procedure was repeated 6x for each Ki-67 stained slide. The index of proliferation was determined for each slide by dividing the number of Ki67-positive cells by the total number of cells counted.
Necrosis is defined by a loss of fine dispersion of chromatin in the cell nucleus, accompanied by fading of distinct cell margins. For xenografts, necrosis is scored as complete, partial (often at the surface), or absent. Infiltration of tumor cells into the CAM is defined by the observation of tumor cells within the mesoderm of the CAM, and is scored as being present or absent.
Three items are scored for host behavior. Fibroblast infiltration is defined as elongated cells leaving the CAM and invading the tumor graft/plaque, and is scored as being present or absent. Vascular ingrowth can be observed as ingrowth of proliferating chick vessels, characterized by the presence of nucleated chick erythrocytes.

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Authors have nothing to disclose.

Time of inoculation and harvest
Timing the day of inoculation was performed using SAOS2 in ECM gel (36 CAMs) and varied between embryonic development day 5 and 10.
Before incubation day 9, the CAM was not consistently large enough to support the ECM gel we applied. At harvest, tumor cells sometimes had to be retrieved from the deeper CAM, and some ECM gel samples were lying loose in the albumen or on the CAM. On days 5 and 6, it was hard to avoid putting the tumor ce...

Sarcoma is a rare tumor of the connective tissues with a high mortality due to therapy resistance1,2. Progress in patient survival is hampered by their low annual incidence, their broad diversity, and the fact that sarcoma cells are reported to be hard to culture in vitro3,4.
The use of cultured cells for preclinical therapy evaluation has revealed that new, apparently active molecules in vitro do not always reflect results in the clinical setting. Furthermore, genome aberrations revealed by gene expression arrays are not always correlated to tumor behavior characteristics in the individual patient5-7. In order to try and solve these problems, personalized medicine has gained in importance, which is reflected in the increased search for xenograft models8-12.
An in vivo assay has the advantage of reflecting the complex interplay between cancer cells and the host tissue environment in solid tumors, necessary for cancer proliferation and invasion13. Currently we study the use of the Chorio-Allantoic Membrane assay (CAM-assay) as a reproducible xenograft model for sarcoma14,15. This assay is widely used for the study of tumor angiogenesis16,17. In literature, however, we have found different protocols for this assay, while other studies observed a marked difference in growth or angiogenesis according to different protocols18,19.
In this article we investigate the effect of varying conditions of the CAM-assay on cell behavior using tumor grafts, tumor-derived single cell suspensions and established sarcoma cell cultures.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent</td>
			<td>Company</td>
			<td>Catalog Number</td>
		</tr>
		<tr>
			<td>Cell Line Nucleofector Kit V</td>
			<td>Amaxa</td>
			<td>VCA-1003</td>
		</tr>
		<tr>
			<td>collagenase 2 solution (500 U/ml RPMI 1640)</td>
			<td>Sigma Aldrich</td>
			<td>C6885</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>41965-039</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D8418</td>
		</tr>
		<tr>
			<td>Dnase solution</td>
			<td>Sigma Aldrich</td>
			<td>DN25</td>
		</tr>
		<tr>
			<td>G418</td>
			<td>Invitrogen</td>
			<td>11811031</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Sigma-Aldrich</td>
			<td>E1270</td>
		</tr>
		<tr>
			<td>mouse primary monoclonal antibody Ki67</td>
			<td>Dako Denmark</td>
			<td>MIB-1</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Fluka</td>
			<td>D76240</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Invitrogen</td>
			<td>20012019</td>
		</tr>
		<tr>
			<td>PBSD</td>
			<td>Invitrogen</td>
			<td>14040083</td>
		</tr>
		<tr>
			<td>peGFP-C1 vector</td>
			<td>Clontech</td>
			<td>632470</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Invitrogen</td>
			<td>15140163</td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Invitrogen</td>
			<td>22409-015</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>Invitrogen</td>
			<td>25300054</td>
		</tr>
		<tr>
			<td>Vybrant cell-labeling DiI</td>
			<td>Lifetechnologies</td>
			<td>22885</td>
		</tr>
		<tr>
			<td>Name of Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
		</tr>
		<tr>
			<td>Countess Automated Cell Counter</td>
			<td>Invitrogen</td>
			<td>C10227</td>
		</tr>
		<tr>
			<td>digital color camera</td>
			<td>Leica</td>
			<td>DFC 340 FX</td>
		</tr>
		<tr>
			<td>Digital Egg Incubator</td>
			<td>Auto Elex Co</td>
			<td>R-COM 50</td>
		</tr>
		<tr>
			<td>FACS</td>
			<td>BD Biosciences</td>
			<td>FACSAriaIII</td>
		</tr>
		<tr>
			<td>Gentlemacs C-Tube</td>
			<td>Miltenyi Biotech</td>
			<td>130-093-237</td>
		</tr>
		<tr>
			<td>Gentlemacs Dissociator</td>
			<td>Miltenyi Biotech</td>
			<td>130-093-235</td>
		</tr>
		<tr>
			<td>Gentlemacs Dissociator User Manual containing h_tumor protocol</td>
			<td>Miltenyi Biotech</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>semipermeable adhesive film (Suprasorb F)</td>
			<td>Lohmann&amp;Rauscher</td>
			<td>20468</td>
		</tr>
		<tr>
			<td>stereo fluorescence microscope</td>
			<td>Leica</td>
			<td>M205 FA</td>
		</tr>
		<tr>
			<td>Tissue-Tek Film automated Coverslipper</td>
			<td>Sakura</td>
			<td>6400</td>
		</tr>
		<tr>
			<td>ultraView Universal DAB Detection Kit</td>
			<td>Ventana Medical Systems Inc</td>
			<td>760-500</td>
		</tr>
		<tr>
			<td>Ventana Automated Slide Stainer</td>
			<td>Ventana Medical Systems</td>
			<td>Benchmark XT</td>
		</tr>
	</tbody>
</table>

the in ovo cam-assay as a xenograft model for sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

Evaluation of the CAM
Tumor grafts become adherent to the CAM (Figure 2A). Single cell suspensions from patient material frequently display a dried, slightly raised plaque (Figure 2D). After excision of the CAM, marked wrinkling of the membrane occurs (Figures 2E and 2F).
For the commercial cell lines the plaque becomes more opaque in time, indicating cell proliferation. Different cell lines in ECM gel...

Watch this Scientific Journal Video about The In ovo CAM-assay as a Xenograft Model for Sarcoma at JoVE.com

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

the-in-ovo-cam-assay-as-a-xenograft-model-for-sarcoma

Research

JoVE Journal

Medicine

25.3K Views.  Ghent University Hospital. Gli In ovo Corioallantoidea membrana (CAM) si innesta con tessuti freschi sarcoma di derivazione tumorale, le sospensioni di cellule singole, e stabilite le linee cellulari del sarcoma fluorescente permanenti e transitori. Il modello è utilizzato per lo studio del trapianto (viabilità, indice di proliferazione Ki67, necrosi, infiltrazione) e host (fibroblasti infiltrazione, crescita interna vascolare) comportamento.

Video: The In ovo CAM-assay as a Xenograft Model for Sarcoma

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma (glioma) and medulloblastoma. The widely used stereotactic approach only allows for the injection of a single animal at a time, is labor intensive and requires highly specialized equipment. The guide screw method, initially developed by Lal et al.,1 was developed to eliminate cumbersome stereotactic procedures. We now describe a modified guide screw approach that is rapid and exceptionally safe; both of which are critical ethical considerations. Notably, our procedure now incorporates an infusion pump that allows up to 10 animals to be simultaneously injected with tumor cells.
To demonstrate the utility of this procedure, we established human U87MG glioma cells as intracranial xenografts in mice, which were then treated with AMG102; a fully human antibody directed to HGF/scatter factor currently undergoing clinical evaluation2-5. Systemic injection of AMG102 significantly prolonged the survival of all mice with intracranial U87MG xenografts and resulted in a number of complete cures. 
This study demonstrates that the guide screw method is an inexpensive, highly reproducible approach for establishing intracranial xenografts. Furthermore, it provides a relevant physiological model for validating novel therapeutic strategies for the treatment of brain cancers.

In order to evaluate novel therapeutic paradigms for the treatment of glioma, physiological relevant models are essential. We utilize an implantable guide screw procedure for establishment of intracranial xenograft models that is more rapid and safer than stereotactic approaches.

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Glutamate and Hypoxia as a Stress Model for the Isolated Perfused Vertebrate Retina

Neuroprotection has been a strong field of investigation in ophthalmological research in the past decades and affects diseases such as glaucoma, retinal vascular occlusion, retinal detachment, and diabetic retinopathy. It was the object of this study to introduce a standardized stress model for future preclinical therapeutic testing.
Bovine retinas were prepared and perfused with an oxygen saturated standard solution, and the ERG was recorded. After recording stable b-waves, hypoxia (pure N2) or glutamate stress (250 &#181;m glutamate) was exerted for 45 min. To investigate the effects on photoreceptor function alone, 1 mM aspartate was added to obtain a-waves. ERG-recovery was monitored for 75 min.
For hypoxia, a decrease in a-wave amplitude of 87.0% was noted (p &#60;0.01) after an exposition time of 45 min (decrease of 36.5% after the end of the washout p = 0.03). Additionally, an initial decrease in b-wave amplitudes of 87.23% was recorded, that reached statistical significance (p &#60;0.01, decrease of 25.5% at the end of the washout, p = 0.03).
For 250 &#181;m glutamate, an initial 7.8% reduction of a-wave amplitudes (p &#62;0.05) followed by a reduction of 1.9% (p &#62;0.05). A reduction of 83.7% of b-wave amplitudes (p &#60;0.01) was noted; after a washout of 75 min the reduction was 2.3% (p = 0.62). In this study, a standardized stress model is presented that may be useful to identify possible neuroprotective effects in the future.

With this study, we introduce a standardized stress model for the isolated superfused bovine retina for future preclinical therapeutic testing. The effect of either hypoxia (pure N2) or glutamate stress (250 &#181;M glutamate) on retinal function represented by a- and b-wave amplitudes was evaluated.

Neuroprotection has been a strong field of investigation in ophthalmological research in the past decades and affects diseases such as glaucoma, retinal ...

The In Ovo Chick Chorioallantoic Membrane (CAM) Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and highly vascularized, making it an ideal system for tumor implantation. Furthermore, the CAM contains extracellular matrix proteins such as fibronectin, laminin, collagen, integrin alpha(v)beta3, and MMP-2, making it an attractive model to study tumor invasion and metastasis. Scientists have long taken advantage of the physiology of the CAM by using it as a model of angiogenesis. More recently, the CAM assay has been modified to work as an in vivo xenograft model system for various cancers that bridges the gap between basic in vitro work and more complex animal cancer models. The CAM assay allows for the study of tumor growth, anti-tumor therapies, and pro-tumor molecular pathways in a biologically relevant system that is both cost- and time-effective. Here, we describe the development of CAM xenograft model of hepatocellular carcinoma (HCC) with embryonic survival rates of up to 93% and reliable tumor take leading to growth of three-dimensional, vascularized tumors.

The In Ovo Chick Chorioallantoic Membrane CAM Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) is immunodeficient and highly vascularized, making it a natural in vivo model of tumor growth and angiogenesis. In this protocol, we describe a reliable method of growing three-dimensional, vascularized hepatocellular carcinoma (HCC) tumors using the CAM assay.

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

Oleic Acid-Injection in Pigs As a Model for Acute Respiratory Distress Syndrome

The acute respiratory distress syndrome is a relevant intensive care disease with an incidence ranging between 2.2% and 19% of intensive care unit patients. Despite treatment advances over the last decades, ARDS patients still suffer mortality rates between 35 and 40%. There is still a need for further research to improve the outcome of patients suffering from ARDS. One problem is that no single animal model can mimic the complex pathomechanism of the acute respiratory distress syndrome, but several models exist to study different parts of it. Oleic acid injection (OAI)-induced lung injury is a well-established model for studying ventilation strategies, lung mechanics and ventilation/perfusion distribution in animals. OAI leads to severely impaired gas exchange, deterioration of lung mechanics and disruption of the alveolo-capillary barrier. The disadvantage of this model is the controversial mechanistic relevance of this model and the necessity for central venous access, which is challenging especially in smaller animal models. In summary, OAI-induced lung injury leads to reproducible results in small and large animals and hence represents a well-suited model for studying ARDS. Nevertheless, further research is necessary to find a model that mimics all parts of ARDS and lacks the problems associated with the different models existing today.

In this article, we present a protocol to induce acute lung injury in pigs by central-venous injection of oleic acid. This is an established animal model for studying the acute respiratory distress syndrome (ARDS).

The acute respiratory distress syndrome is a relevant intensive care disease with an incidence ranging between 2.2% and 19% of intensive care unit ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...