The overall goal of this experimental method is to visualize and quantitate the dynamic process of vasculogenesis over time. This method can help answer key questions in the fields of angiogenesis and vasculogenesis about the rates of vessel formation and the differences between formed network structural patterns. The main advantage of this technique is that it enables the quantification of large numbers of vasculogenesis time points, providing an opportunity to assess the kinetics of the vasculogenic process.
This method can be applied to any cell population or pathologic state that impacts the processes of vasculogenesis or angiogenesis for the study of individual cell populations or co-culture systems. Approximately one to two hours before beginning the experiment, preheat a Microscope Stage Top Incubator to 37 degrees Celsius, and set the carbon dioxide to 5%and the humidity to 85%Fill the incubator's humidity reservoir if it is empty. Place the live-cell chamber onto the microscope, and then, place an empty chambered slide into one of the two open positions within the live-cell chamber.
Fill the slide with distilled water and set a secondary blower to 39 to 42 degree Celsius to heat the slides from below and to minimize condensation. Maintaining the proper temperature and humidity within the live-cell imaging chamber is critical to the success of the experiment. Add a second slide as a blank until the experimental slide can be added.
Then, add water to the reservoirs surrounding the wells on the second slide to mimic the conditions during the imaging experiment. To load the experimental chamber slide with basement membrane matrix, first load a 20 microliter pipette with a cold 20 microliter pipette tip and use scissors to cut approximately one centimeter from the end of the tip. Set the pipette volume to slightly greater than 10 microliters and slowly aspirate the matrix into the modified pipette tip.
Immediately place the pipette tip into the center of one slide well, gently touching the tip to the bottom of the well, and slowly depress the plunger to push the matrix out. After adding matrix to all 15 wells, push the lid all the way onto the slide. Accurate and consistent pipetting of the Matrigel volumes into each well is critical for obtaining high quality phase contrast images.
Then, place the slide at 37 degrees Celsius for 30 to 60 minutes next to a 50 milliliter conical tube cap filled with two to three milliliters of PBS to reduce matrix dehydration. When the matrix has solidified, remove the supernatant from an overnight-cultured endothelial colony-forming cell culture, and wash the cells with seven milliliters of PBS. Aspirate the PBS wash and detach the cells with two to three milliliters of Trypsin at 37 degrees Celsius for two to five minutes.
Then, resuspend the dissociated cells in three milliliters of Endothelial Growth Medium-2 to break up any cell clumps and transfer the resulting cell suspension into a 15 milliliter conical tube. After all of the endothelial colony-forming cell samples have been resuspended, pellet the cells by centrifugation and resuspend the pellets in one to two milliliters of fresh Endothelial Growth Medium. Count the number of viable cells in each sample and add enough endothelial colony-forming cells into a new 1.5 milliliter tube to allow dilution of the cells to a 1.4 times 10 to the fourth cells per 175 microliters of medium concentration.
Then, mix each master mix with gentle pipetting. Remove the slide lid and cede 50 microliters of each master mix per well in triplicate onto the slide. To image the cells, replace the blank slide with one experimental slide and add water to the reservoirs surrounding the cells on the slide.
Cycle through each stage position and confirm that the center of the first well is selected. Focus on the cells plated in the well and set the Z position of the stage. When the settings are accurate, turn out the lights to begin the imaging experiment.
When the imaging is complete, scale and export image stack files within the Image Capture software, saving the images as TIFF files for analysis. To analyze the images, drag the Kinetic Analysis of Vasculogenesis file from its folder into the gray bar at the bottom of the software window to open the software, and click Save to add the software to the list. Click File and Open to select the image file to open, or drag the image file to the gray bar within the software.
Click Image, Adjust, and Brightness/Contrast to open the Brightness/Contrast window. Click Reset within the Brightness/Contrast window to visualize the image. Then, click Image, Type, and 8-bit, to convert the images to 8-bit.
Select Tools and Region Of Interest Manager, and use an appropriately shaped selection tool to create a new region of interest. Click Add to add the region of interest shape to the Region Of Interest Manager window and click on the Region Of Interest label in the Region Of Interest Manager to view the region on the image. Adjust the region of interest as necessary.
When the region is in place, click Image, then Crop, to crop the image. Then, open the Edit menu and select Clear Outside to delete the image data outside of the region of interest for every image in the stack. Next, open the Plugins menu and select the Analyze Network plugin.
Use the drop-down arrow in the popup window to change the Thresholding Method and click OK to initiate the software processing. At the end of the analysis, a data table and a stack of fused images depicting Skeleton and Mask renditions will open. Click File and Save As to transfer the numerical values into a spreadsheet, and save the spreadsheet containing the raw data values.
Click on the fused Skeleton and Mask image and save the fused Skeleton Mask image stack and the cropped phase contrast image stack as TIFF files. Then, click on the Region Of Interest Manager window, select the region of interest used to crop the images, and click More, and Save, to save the region of interest for future use. Endothelial colony-forming cell networks, identified by Kinetic Analysis of Vasculogenesis, are represented pictorially as Skeleton and Mask renditions to illustrate the structures used by the software for quantification.
When the quality of the phase contrast images is high and sufficient contrast is achieved, Kinetic Analysis of Vasculogenesis will accurately identify endothelial colony-forming cell networks as indicated by the similarities observed between the phase contrast image and the Kinetic Analysis of Vasculogenesis generated Skeleton and Mask renditions. If phase contrast images do not have high contrast, or if imaging artifacts such as gridding occur, the network detection accuracy is reduced and the outcomes become ambiguous. However, different thresholding methods, such as Mean and Otsu, shown here, can be selected prior to analysis in the software drop-down menu to accommodate differences in image quality.
Using Kinetic Analysis of Vasculogenesis to determine if gestational diabetes mellitus exposed endothelial colony-forming cells display altered kinetics of network formation reveals heterogeneously altered network structure formation in gestational diabetes mellitus cultures compared to endothelial colony-forming cell cultures from an uncomplicated pregnancy. Indeed, uncomplicated pregnancy samples typically form a greater number of closed networks compared to gestational diabetes mellitus samples. All of the endothelial colony-forming cell samples, however, display a biphasic pattern of network formation overall, although the rates of formation and the maximal number of networks achieved varies across the samples.
Following this procedure, the data values generated by Kinetic Analysis of Vasculogenesis can be graphed and analyzed to determine if the network structures formed by the different samples are statistically different, and if so, at which time points. Kinetic Analysis of Vasculogenesis enables the efficient processing of time-lapse experiments involving a large number of images. Once mastered, the image acquisition, post-processing and analysis can be completed in two days if all of the systems are performing properly.
