The overall goal of this procedure is to observe and analyze invasive migratory behaviors in primary human glioblastoma cells. This method can help answer key questions in the field of neurooncology, such as what are the phenotypic and molecular properties underlying patient-specific differences in disease progression and treatment responses? The main advantage of this technique is that it allows the direct manipulation of primary cells while preserving the in vivo tissue architecture and cellular heterogeneity of glioblastoma.
Begin by using sterile forceps to place empty PTFE culture inserts into each well of a six-well plate containing one milliliter of slice culture maintenance medium per well. When all of the inserts have been added, place the plate in a humidified water-jacketed tissue culture incubator at 37 degrees Celsius and 5%CO2. Next, place the tumor tissue pieces in a Petri dish containing ice-cold processing medium, and use a pipette to gently wash the tissue three times with fresh medium to remove any adherent red blood cells.
After the third wash, use the scalpel to cut the tumor pieces into approximately three by three by 10 millimeter strips, carefully removing any attached vessels and avoiding necrotic or cauterized tissue. When all of the tissue has been trimmed, add five to seven millimeters of 37 degree Celsius liquid agarose into a two-cubed centimeter plastic embedding mold, and chill the agarose on a bed of ice for one minute. Place two to four strips of tissue into the agarose with the long axis oriented vertically, leaving the tissues on ice for another two to five minutes after their placement.
With the agarose has solidified, cut the sides of the form with a scalpel to gently remove the agarose from the mold. And use a generous drop of cyanoacrylate glue to fix the agarose block to a vibratome specimen plate. When the glue has set, submerge the agarose block in ice-cold processing medium and use a trimmed sterile plastic pipette to bubble a 5%CO2, 95%O2 mixture into the medium within the vibratome reservoir.
Set the vibratome slice thickness to 300 to 350 microns, and adjust the blade advance speed and blade amplitude according to the tissue consistency and vibratome specifications. Obtain the appropriate number of slices according to the planned experimental analysis, using a stainless steel micro spatula to transfer the slices into a Petri dish containing ice-cold processing medium as they are acquired. When all of the slices have been acquired, use the micro spatula to transfer each slice onto a single PTFE insert.
After a 12 to 24 hour incubation, equilibrate a new six-well plate containing fresh slice culture maintenance medium in the cell culture incubator for 15 minutes. Then use sterile forceps to grasp each insert by the rim and transfer the inserts into individual wells of the new six-well plate. Between seven to 10 days of culture, gently add five to 10 microliters of GFP-expressing retrovirus dropwise onto the surface of each tissue slice and return the plate to the incubator.
When the signal is apparent, equilibrate one milliliter of fresh slice medium in a glass-bottom dish and use sterile forceps to transfer the inserts into the dish. Place the dish into a 37 degree, 5%CO2 sealed microscope stage top incubator of a confocal microscope. Use a long working distance, 10x air objective in conjunction with single or multi-photon imaging to capture three-dimensional time-resolved images of cell migration.
Visualize the slice with the microscope, locating a suitable field with an adequate density of fluorescently-labeled tumor cells between the slice edge and the center of the tissue. Then in the multidimensional analysis mode of the imaging software, set the center of the Z-stack such that all of the positions to be imaged contain a visible fluorescent cellular signal, and set an adequate time for each Z-stack acquisition. To begin, open a Z-stack file in ImageJ and select Image, Stacks, and Z Project.
Select the first and last time frame of the Z-stack for analysis, and choose Max Intensity as the projection type to generate a maximum intensity projection or MIP rendering. To generate MIPs for each time point in the series at once, check the box All time frames. Create an MIP from each set of Z-stack images captured at each region imaged.
Open MTrackJ from the Plugins menu, and click the Add button. To manually identify the cell body centroid in the tumor cell of interest, click on the visually approximated center point of the cell. Demarcate the cell body location in each timeframe of the series of images to create a unique track for each cell, sequentially marking the cell body locations of the next cell.
Once a population of cells has been tracked in a given tumor microregion, use the measure function to export all of the cell track coordinates and save the files in the xls format for later analysis. Finally, use the coordinates recorded for each point along a cell's migration track to perform quantitative analyses of the tumor invasion. Organotypic glioblastoma slices demonstrate a concordance with the originally tumor tissue throughout the culture, including the maintenance of pseudopalisading necrosis and microglia for up to 15 days.
Under hypoxic conditions, the slices mount a rapid physiologic response, inducing the release of vascular endothelial growth factor into the medium, a process that occurs abundantly within the glioblastoma microenvironment in vivo. The quantitative measurement of time-lapsed image GFP-expressing tumor cells allows the calculation of the migration speed and the directionality of the glioblastoma cells across tumor regions in samples. Tracking the migration of intermingled but morphologically distinct motile tumor cells in microglia within a slice culture reveal cell type-specific patterns of movements.
Imaging the slice regions approximately every 10 minutes also provides an adequate temporal resolution for recording time lapse images of the tumor cells undergoing cell division. For example, here are a pair of dividing cells that were captured as they paused for migration, completed mitosis, and reinitiated migration in their daughter cells without delay, all within a three-hour time frame. Once mastered, this technique can be completed in 90 minutes if it's performed properly.
