The overall goal of this experiment is to quantitatively characterize angiogenic and non-angiogenic phenotypes of human osteosarcoma cells in a systematic manner combining analyses of cell morphology, proliferation, and motility. This method can help answer key questions with respect to human cancer establishment and progression. In particular, the escape from tumor dormancy and the endogenic switch.
The main advantage of this technique is that multicellular parameters can be analyzed simultaneously by this integrated, high-spread method. This includes cell area, cell thickness, cell volume, proliferation rate, doubling time, motility speed, migration, and motility. To begin, prewarm the culture medium to 37 degrees Celsius in a water bath.
Then remove cryogenic vials of angiogenic and non-angiogenic human osteosarcoma cells from the liquid nitrogen tank. Immerse the bottom of the vials into a 37 degree Celsius water bath and gently shake the cryogenic vials to accelerate the thawing process. As soon as cells are thawed, spray the vial with 70%ethanol and wipe the vial to sterilize it.
Then immediately aspirate the cell suspension from the vial and gently suspend them in 10 milliliters of warmed culture medium. Next centrifuge the cell suspensions for five minutes to pellet the cells. Back in the hood, remove the supernatant and re-suspend the cell pellet with 10 milliliters of warmed culture medium.
Then transfer the cell suspension into a T75 flask. Gently pipette the cell suspension several times to evenly disperse the cells. Place the flask into an incubator and allow the cells to attach for at least 12 hours.
Change the culture medium every two to three days. When the cells reach 80 to 100 percent confluence, remove the culture media from the flask and wash the cells with five milliliters of sterile PBS without calcium and magnesium. Then add one milliliter of a 0.25%tripson EDTA solution into the flask and briefly swirl the flask.
Incubate the flask for two to three minutes and then check the cells under a microscope to make sure the cells are rounded up and detached. Once the cells are detached, use a pipette to add 10 milliliters culture medium and gently pipette the medium up and down several times to break up any remaining cell aggregates. After counting the cells, seed two million of them into a new T75 flask.
Place the flask in an incubator and grow the cells to between 80 and 100 percent confluence before seeding the cells for the quantitative phase imaging experiment. Harvest cells by tripson digestion and seed angiogenic and non-angiogenic human osteosarcoma cells in six well plates at the density of 50, 000 to 300, 000 cells per well. Cover the cells with a total of five milliliters of culture medium.
Then incubate the plates for at least 12 hours to allow time for the cells to attach. Replace the medium with fresh media before imaging. Clean a cover slip by rinsing it several times with running deionized water.
Then immerse it in a 75%ethanol solution for 15 minutes. Place the sterilized cover slip into a laminar flow hood to dry. Next carefully place the cover slip into a six well plate, avoiding obvious bubbles.
Place the plate into the incubator to equilibrate for at least 15 minutes. If fog forms on the lid of the plate, use a sterile cotton swab to wipe it clean prior to imaging. Open the quantitative phase imaging software and initialize the system.
Make sure that the values are acceptable in the self-calibration of exposure time, pattern contrast, and hologram noise. Next, place the six well plate onto the stage of the quantitative phase image microscope. Back in the software, click on live capture.
Then go to the software focus menu and select manual. Next go to the microscope setting menu and coarsely focus the phase images of the cells by adjusting the work distance to obtain outlines for cells in phase images. When finished, go back to the software focus menu and change the focus setting to automatic.
Now click on new experiment. Begin selecting image acquisition positions using the mouse or the arrow keys on the number pad. Click remember after each selection.
Once the positions have all been selected, go to the time lapse section and set up the imaging intervals so that they are shorter than five minutes and the total time period is 48 hours. Start the experiment by clicking capture. This will automatically focus and acquire images at the selected positions at each interval time points.
Following image acquisition, analyze the morphology of the cells by first clicking on identify cells. Adjust the numeric setting for the background threshold so that cell areas are well separated from the background noise. Next adjust the setting number for object size to make sure that each cell has only one nucleus.
Manually modify any cell segments not matching this requirement by using manual changes. Then use the analyze data function in the software to analyze cells. Start a new data analysis by clicking new analysis.
Drag selected images into the source frames tab and record cell morphology parameters, including cell area, optical thickness, and volume for each individual cell. When finished, choose the scatter plot or histogram mode and export the data as tables or figures. Select at least five images spaced 12 hours apart and record cell numbers that are provided by the software.
Estimate the proliferation rate of the cells by normalizing with the initial cell numbers and then plotting the cell numbers. Finally, estimate the doubling time by fitting the data points using an exponential growth curve. To analyze cell motion use the track cells function in the software.
Start by clicking on new analysis to start a new data analysis. Then drag a series of images from the time period of interest into the source frames tab. Next, under the add cells selection mode, randomly choose 10 to 30 cells by clicking on them.
Be sure to review the tracking in the series of images. In the event that the system loses track of a cell or tracks the wrong cell, manually adjust the tracking cell by clicking on identify to identify cells or by going to the select mode and clicking on modify location to modify a cell's location. When finished go to plot movement and choose rows plot of cell trajectories or go to plot features and plot for the other motion related parameters, such as motility speed, motility, migration, and migration directness.
Export the data as tables or figures. In this protocol, quantitative phase images are presented as holographs. The cells shown here depict the typical cell morphology for angiogenic and non-angiogenic human osteosarcoma cells.
The cell thickness is calculated by the refractive index and optical path length and is shown here as a scatter plot. The two difference cell pheno types displayed different distribution patters and indicate that the angiogenic cells have smaller cell areas and a greater cell thickness than the non-angiogenic cells. The cell proliferation rate of the two cell types can be calculated from 2D images such as these, which have been segmented and span the course of two days.
For these specific cell types, no significant differences were found. To determine cell motion characteristics for the cells, individual cells must be selected and tracked over time. Here the tracked cells are outlined with different colors.
Each individual cell was tracked over the course of four hours with their total path trajectory described here where the zero, zero point is their starting location and their final location is indicated by the colored dot. After watching this video you should have a good understanding of how to use quantitative phase imaging to quantitatively compare a pan of cell morphological and behavior parameters. In this case, between angiogenic and non-angiogenic cell phenotypes.
After this development, this technique paved the way for researchers in the field of cancer research to explore the escape from tumor dormancy and the role of angiogenecy in this process. In a continuous and non-labeling platform. Don't forget that working with human cell lines can be extremely hazardous and precautions such as wearing personal protective equipment should always be taken while performing this procedure.
