Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds

The overall goal of this procedure is to offer a relevant organ culture method for studying tumor invasion in three dimensions. This is accomplished by first preparing normal tissue fragments obtained from embryonic chicken hearts. In the second step, aggregates of tumor cells are prepared in suspension.

The tumor cell aggregates are then individually confronted with the normal tissue fragments on a semi-solid agar bed for incubation in a suspension culture. In the final step, the confronting cultures are fixed for paraffin embedding and histological sectioning. Ultimately, the invasion of the tumor cells into the tissue fragments can be visualized by serial microscopic analysis of the tissue sections.

The main advantage of this technique over existing methods like invasion assays with gels is that with this technique, tumor cells are confronted with three dimensional living tissue fragments. The implications of this technique extend to the therapy of cancer as there is an unmet need for anti invasive drugs. Generally, individuals new to this method will struggle, and the reason is that they disregard the importance of using the specific dimensions of the tissue fragment and of the tumor cell aggregates.

Though this method can provide insight in cancer invasion, it can also be applied to other systems. For instance, studying invasion of peripheral tissues by inflammatory cells or the integration of stem cells into cardiac tissue. Supreme culture, the chick heart fragments begin by incubating a fertilized egg at 37 degrees Celsius for nine days.

On day nine, disinfect the shell with 70%ethanol. Then in a tissue culture cabinet, use blunt forceps to open the shell. Next position an enucleation spoon around the neck of the embryo and pull the embryo out of the shell.

Place the embryo in a five centimeter glass Petri dish containing five milliliters of ringer salt solution. Then holding sharp forceps in each hand. Rip open the ventral thoracic skin, remove the sternum and use iridectomy scissors to dissect out the heart and disconnect its major blood vessels.

Transfer the heart to a new five centimeter glass Petri dish containing five milliliters of culture medium. Supplemented with 5%fetal bovine serum under a microscope with a calibrated ocular grid. Using microdissection scissors resect the upper cranial third of the heart to remove the atria and associated vessels.

Then use sharp forceps to dissect the pericardium from the ventricles. Next, make a sagittal hemi section in the ventricles and remove the blood. Then transfer the ventricles into another five centimeter Petri dish with five milliliters of fresh culture, medium and fetal bovine serum, and cut the tissue into 0.4 millimeter diameter pieces.

When all of the ventricles have been cut, rotate the dish gently to drive all of the fragments towards the center of the dish. Then use a stainless steel needle to move all of the Cora Eliana towards the periphery of the Petri dish. Now use a glass pasture pipette to transfer the heart fragments into a 50 milliliter Erla Meyer flask covered with a thin film of culture.

Medium gas the flask with 5%carbon dioxide and incubate the fragments on a gyratory shaker at 37 degrees Celsius with 70 revolutions per minute for 24 hours. The next day, transfer the fragments into a Petri dish and use a needle to discard the Corporal Eliana, the dark necrotic fragments, and any tissue conglomerates. Incubate the fragments in a 50 milliliter erlenmeyer flask containing six milliliters of culture medium on the gyratory shaker for another 60 hours, two and a half days later, use the microscope and needles to select the steroidal fragments that exhibit a thin homogenous layer of fibroblastic cells and a diameter of 0.4 millimeters.

Then transfer the pre cultured heart fragments to another Petri dish containing fresh culture medium. Three days before the start of the confronting culture, suspend 100, 000 test cells per milliliter in six milliliters of the appropriate culture medium in a 50 milliliter erlenmeyer flask. Then incubate the flask on a gyratory shaker at 37 degrees Celsius and 70 revolutions per minute with the appropriate percent of carbon dioxide.

After 72 hours, view the aggregates under the microscope and select the steroidal cell aggregates with a diameter of 0.2 millimeters. Next, transfer eight of the 0.4 millimeter pre cultured heart fragments suspended in a minimal amount of culture medium into an embryological. Watch glass containing semi-solid agar medium.

Move the fragments into a circle and use a small piece of filter paper to aspirate the excess medium. Place 10 of the 0.2 millimeters steroidal cell aggregates into the circle. After removing the excess medium, move one aggregate towards each of the pre cultured heart fragments until they make contact with each other.

Now, seal the lid of the watch glass with paraffin and incubate the co culture at 37 degrees Celsius for four to 24 hours depending on the adhesive properties of the test cells to the pre cultured heart fragments. At the end of the incubation, immerse a confronting pairs in 37 degrees Celsius prewarm culture medium, and use a pasture pipette to transfer each pair into individual five milliliter Erla Meyer flasks containing 1.5 milliliters of culture medium each Then incubate the flasks on the gyratory shaker at 37 degrees Celsius and 120 revolutions per minute with the appropriate percent of carbon dioxide passed through. Two supplemental five milliliter erlenmeyer flasks filled with two milliliters of ringer salt solution for histological analysis of the confrontations.

At the end of the incubation period, transfer the cultures to ringer salt solution for a few seconds to remove the serum proteins. Next, transfer the pears to individual three centimeter Petri dishes containing three milliliters of Bowen Holland solution. After two hours, rinse the cultures three times in three milliliters of demineralized water before incubating them in tap water for two hours to remove as much fixative solution as possible.

Finally, transfer the pairs into three milliliters of 70%ethanol each for up to several days. In these images of successful cultures, no immune reaction by the normal tissue can be observed in either the tumor or heart tissue, confirming that the chick embryos were used before the development of the immune rejection system. There are also no bacteria visible indicating an absence of gross contamination during the culture period.

Further, the rounded periphery of the sections confirms that the cultures existed in suspension without signs of any temporary adherence to the erla mire flask wall. Many compounds have been tested with this Chick heart invasion assay. For those that were structurally related, a quantitative structure activity relationship was established based on results of the assay with the data demonstrating a clear correlation between the predicted and experimental anti invasive activities.

Indeed, this confusion matrix can be used to summarize the weaknesses and the strengths of the assay, giving a rough expression of the accuracy and the reproducibility of the experiment. While attempting this new procedure, it's extremely important to stick strictly to the prescribed dimensions of the tissue fragments Following this procedure. Other methods like confrontation with non-cancer cells can be performed to answer additional questions like Do inflammatory cells invade?

After watching this video, you should have good understanding of how to discriminate between invasive and non-invasive tumor cells. Don't forget that working with fixatives can be extremely hazardous and precautions such as air ventilations should be taken while performing this procedure.