This procedure demonstrates how to measure the strength of intracellular cohesion in 3D tissue like aggregates. The first step is to generate spherical tissue like aggregates and to transfer these into a tissue culture medium filled petri dish. Next individual aggregates are aspirated into a pasta pipette and loaded into the lower compression plate of the tensiometer chamber.
Then the upper compression plate is connected to a nickel chromium wire, which connects it to the electro balance and establishes a zero baseline weight referred to as F zero. The experiment is initiated by raising the lower compression plate until the aggregate becomes compressed between the lower and upper compression plates and is terminated when aggregates reach, shape and force equilibrium or FEQ. The final steps of the procedure are to capture images, measure aggregate geometry and force of resistance, and to input these measurements into the young LA place equation.
Ultimately, results can be obtained that show changes in tissue cohesion and how these could influence tumor dispersal or spatial positioning between different cell populations through aggregate spreading assays or cell sorting assays respectively. So this method can help answer key questions in the fields of tumor biology, embryonic development, and tissue engineering. Questions such as How does tumor cohesion influence malignant invasion, the stratification of germ layers during gastro?
Or how can we actually engineer cohesion to generate functional organ like structures In vitro Experimental manipulations involving the culture of living cells should be performed under aseptic conditions in a designated lamina flow hood. Sterile solutions and sterile LabWare should be used throughout. To begin this experiment, harvest adherence cells at 90%co fluency.
First, aspirate the culture media and rinse the cell sheet twice with PBS. Then aspirate the PBS and pipette two milliliters of 0.05%trips in EDTA solution onto the monolayer. Place the plate into the 37 degrees Celsius tissue culture incubator and incubate until cells detach.
Next, remove the plate from the incubator and halt ization by adding two milliliters of complete medium. Use the five milliliter pipette to tri rate the mixture until the cells are fully in suspension. Then transfer the cell suspension into a 15 milliliter conical tube.
Pipette 40 microliters of a 10 milligram per milliliter, DNA solution into the cell suspension and incubate at room temperature for five minutes Following incubation, vortex the cell suspension briefly and then centrifuge at 200 times gravity for five minutes. Once the centrifugation is complete, aspirate the sup, natant and discard. Then Reese, suspend the pellet in one milliliter of complete medium and repeat the centrifugation to wash Reese.
Suspend the pellet in one milliliter of complete medium, then perform a cell count and if necessary, adjust the concentration of the cell suspension to one time. Center the six cells per milliliter with complete medium pipette. Three milliliters of the cell suspension into a 10 milliliter round bottom flask and incubating a shaking water bath at 37 degrees Celsius and 5%carbon dioxide for four hours at 120 rotations per minute until the cells recover from ionization as indicated by the cells sticking to one another to form tiny clusters.
Next, transfer the cells to a glass round bottom centrifuge tube and centrifuge at 200 times gravity for four minutes to form a thin sheet following centrifugation. Loosen the cap of the tube to allow gas exchange and then place the tube in the tissue culture incubator. Incubate for 24 hours, where upon the sheet should become coherent.
After 24 hours, gently swell the centrifuge tube to dislodge the sheet. Then pour the contents of the culture tube into a small sterile glass tissue culture dish. Use micro scalpels to cut the cell sheet into fragments of various sizes and then transfer the fragments to a 10 milliliter round bottom flask with three milliliters of tissue culture medium.
Incubate the fragments at 37 degrees Celsius in the shaking water bath at 120 rotations per minute under 5%carbon dioxide for two to three days or until they becomes spherical. Aggregate formation may also be performed using the hanging drop method. According to the previously published protocol, the tissue surface TENSIOMETER or TST setup consists of the main tensiometer chamber containing the compression cell, a water bath for maintaining the temperature of the chamber, A dissecting microscope with an attached CCD camera, a car electro balance with power supply, and a chart recorder.
The compression cell of the TST is composed of two chambers, an outer chamber and an inner chamber. The inner chamber houses the poly two hydroxyethyl methacrylate coated upper compression plate and lower compression plate. First, set the circulating water pump to 37 degrees Celsius and ensure that the pipes are connected to the ports on the outer chamber.
Then screw the lower assembly, which is used to elevate the aggregate to initiate compression into the base of the inner chamber. Next, prepare a length of flame straightened nickel chromium wire by first hanging a 15 inch length of wire from a retort stand and clamping a small binder clip to the end. Then run a bunts and burner up and down the length of the wire until the wire glows red.
Use wire clippers to cut the straightened wire to the appropriate length. Then use two razor blades to bend the wire approximately one quarter of an inch from the end to form a small hook. Finally, fill the inner chamber of the TST with carbon dioxide independent tissue culture medium that has been degas by incubation at 37 degrees Celsius for 24 hours.
Usepa pipette to displace any air bubbles that may have formed on the lower compression plate. The TST apparatus is now ready for the aggregate compression experiment. Use APA pipette to aspirate a cell aggregate of 200 to 300 microns from the culture plate.
Then gently expel the aggregate onto the lower compression plate in the inner chamber of the TST apparatus. Position the aggregate as close to the middle of the lower compression plate as possible by gently nudging it with a capillary tube equipped with a small hair loop. Connect the nickel chromium wire up a plate assembly to the balance arm of the can venton Model 2000 recording electro balance.
Adjust the position of the balance until the upper and lower compression plates are parallel and are directly above one another. Allow the upper plate to settle. Then zero the chart recorder to the established baseline weight.
Use the dissecting microscope equipped with CCD camera to capture an image of the uncompressed aggregate. Next, adjust the height of the inner core apparatus to control the desired degree of compression and then raise the lower compression plate until the aggregate is compressed against the upper compression plate. Use the dissecting microscope equipped with CCD camera to monitor the compression and to capture images of the aggregate such as the one shown here.
Continuously record the apparent upper compression plate weight change on a strip chart recorder throughout the compression until achievement of shape. Equilibrium is denoted by the leveling off of the cans balance voltage output. Once again, use the dissecting microscope equipped with CCD camera to capture an image of the compressed aggregate.
This image will be used to measure aggregate geometry After capturing the image, lower the compression plate to decompress the aggregate to measure the compressive force at equilibrium. Read the chart recorder tracing as the difference between the force measured just prior to decompressing the aggregate and the established zero baseline. Repeat the process using a different degree of compression to determine force equilibrium this time.
Measure the compressive force and geometry at force equilibrium and apply these measurements along with the measurements for shape equilibrium to the Young laplace equation to generate numerical values of apparent tissue surface tension. According to the instructions in the written protocol, this table shows tissue surface tension measurements and confirmation of aggregate liquidity for aggregates of rat prostate, fibroblasts and smooth muscle cells. Aggregates of rat prostate fibroblasts cells have a surface tension of 22.8 plus minus 1.1 dines per centimeter.
Moreover, the mean surface tension values measured after compression one and after compression two were statistically identical when compared by a paired T test. We also compared the ratios of sigma two over Sigma one and of F two over F1 to ensure that these aggregates did not obey hook's law as they would if they behaved as elastic solids as demonstrated in table one. The ratio of sigma two over Sigma one does indeed approach one.
Moreover, the ratio of F two over F1 was significantly greater than sigma two over sigma. One Further confirming that these aggregates do not obey hook's law and in fact behave as liquids in contrast, as is evident in table one, the ratio of sigma two over sigma one of rat prostate smooth muscle cells is significantly greater than one and was not statistically different than that of F two over F1.To further demonstrate liquid like behavior, we explored the relationship between surface tension and aggregate volume as can be seen here. Volume is independent of sigma for RPF cells as depicted by the red regression line, whereas there appears to be some dependence of sigma on volume for rat prostate smooth muscle cells as depicted by the blue regression line.
These data further confirm that rat prostate fibroblast aggregates behave in a liquid like manner, whereas those of rat prostate smooth muscle cells appear to behave more as elastic solids. So after its development, this technique paved the way for researchers in the field of embryonic development, malignant invasion and tissue engineering to explore the role of tissue cohesion in establishing compartments and boundaries between different cell types during embryonic germ layer development, during tumor stromal cell interaction and pancreatic eyelet cell rearrangement in a number of different animal models including amphibians, telio, sine avian, and human models.
