制备及分析

The overall goal of this set of procedures is to generate three-dimensional cell cultures to function as in vitro breast cancer surrogates, and to present a method for measuring cell growth in histologic sections of the surrogates. Three-dimensional cell culture is a more physiologically relevant method of modelling cell behavior in vitro than is two-dimensional cell culture. By altering cellular composition in experimental conditions, this set of procedures can be adapted to address a variety of biological questions, and to assess the efficacy of candidate therapeutics.

To incorporate the cells into the ECM, on ice, add the cells and the first four related components listed in table one in the accompanying manuscript, into a two millimeter microcentrifuge tube. Next, gently mix them by pipetting, and avoid forming any bubbles. Monitor the pH level using the phenol red in the 10XD MEM to make sure that the mixture shows an orange-pink color, indicating a pH of seven.

If the mixture appears yellow and the pH is too low, slowly add 7.5 percent sodium bicarbonate one drop at a time until the appropriate color is reached. Remember to keep the mixture on ice, and work quickly to prevent premature ECM polymerization. For solid 3D cultures keep the chamber slide on ice to prevent premature ECM polymerization.

Slowly pipette 100 micro-liters of the cell ECM mixture into each well of the eight well chamber slide, and pipette the cell ECM mixture around the edges of the well to help better distribute the mixture. Next, incubate the surrogates at 37 degrees Celsius, five percent CO2, for 45 minutes to allow ECM polymerization. After forty-five minutes add 100 micro-liters of the culture media to each well, and incubate at 37 degrees Celsius and five percent CO2 for the duration of the experiment.

Change the culture medium every two days. A limitation of 3D culture is that the diffusion of oxygen and nutrients into the volume may inadequate to support cell viability. The addition of a bio-reactor system may help to overcome this limitation and enhance growth.

To profuse 3D cultures in a bio-reactor system, sterily prepare and assemble the portion of the bio-reactor system that houses the surrogates. In a cell culture-hood, using a syringe with a 26 gauge needle, inject the cell ECM mixture into the area of the perfusion bio-reactor designed to contain cells, and quickly proceed to the next step. To ensure more even distribution of cells within the surrogates, place the bio-reactor component housing the surrogates into a 50 milliliter conical tube.

Continuously rotate the surrogates at 18 rpm while incubating at 37 degrees Celsius in an in situ hybridization oven or an incubator for 45 minutes to allow ECM polymerization. Following polymerization and the addition of medium connect the bio-reactor assembly to the pump. Start medium perfusion in an incubator at 37 degrees Celsius, five percent CO2.

Continue medium perfusion for the duration of the experiment, and change the culture medium as required for the specific experimental design. At the end of the experiment encase the surrogates in specimen processing gel to keep the surrogates intact during processing, and facilitate histologic sectioning. To do so, melt the specimen processing gel in a 60 degree Celsius water bath until ready to use.

Then, pipette approximately 300 micro-liters of specimen processing gel into the bottom of a labeled cryomold. Using a scalpel blade and forceps, carefully remove a surrogate from the bio-reactor or from the well of an eight-well chamber slide, and place it into the cryomolds containing specimen processing gel. Pipette approximately 300 micro-liters of specimen processing gel to cover the surrogate in the cryomold.

Then, incubate it at four degrees Celsius for 30 minutes. Once the specimen processing gel has solidified, remove the specimen processing gel containing the surrogate from the cryomold and place it into a tissue cassette. If more than one surrogate is to be put into the same tissue cassette, tissue marking dyes can be used to distinguish samples following fixation and processing.

Subsequently, place the tissue cassette containing the surrogate into ten percent neutral buffered formula for ten to twelve hours at room temperature to allow complete fixation. Following fixation, transfer the tissue cassette containing the surrogate to 70 percent ethanol. The fixed surrogate is now ready for processing to paraffin and subsequent histologic sectioning.

To measure the cell density and photo-microscopic images of H and E stained sections, open an image file of the surrogate. Using the Polygon tool in ImageJ and the edges of the ECM as a guide, outline the area of the surrogate by dragging the mouse and clicking to make anchor points. Once outlined, select Measure under the Analyze tab.

Next, upload the image files used to measure the surrogate area into Cell Profiler by dragging the image files to the file list. Then, assign a name to each image imported in the Names and Types Import module and Select the image type. After the analysis pipeline is generated, start Test Mode and click Run in Cell Profiler.

And evaluate filtered objects to ensure that the majority of the nuclei in the test image are appropriately identified, and the optimal parameters are chosen to identify the cells. The nuclei included will be circled in green. Then, save the project, exit test mode, and click Analyze Images.

Shown here is a representative image of the H and E stained section from a perfused surrogate grown for seven days. And this is a representative image of the H and E stained section from a solid surrogate grown for seven days with medium changed every two days. The cellularity is much lower in the solid surrogates.

This graph shows the comparison of the mean number of nucleated cells per area following seven days of growth. A perfused and solid surrogate. These numerical data support the cellularity in the photo micro-graphs of the H and E stained sections.

And this graph shows the comparison of Ki-67 labeling index, which is the meaure of cell proliferation following seven days growth of perfused and solid surrogates. These data again support greater growth in perfused surrogates. Following this procedure, other methods such as in situ hybridization and amino histochemistry can be performed on histologic sections of the surrogates to answer questions like RNA and protein expression.

After watching this video, you should have a good understanding of how to prepare and process three-dimensional in vitro tissue surrogates, and perform surrogate analysis on H and E stained histologic sections.