Generating and Maintaining Slice Cultures from Human Glioblastoma Tissue
Transcript
Begin by using sterile forceps to place empty PTFE culture inserts into each well of a six-well plate containing 1 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 a scalpel to cut the tumor pieces into approximately 3-by-3-by-10 millimeter strips, carefully removing any attached vessels and avoiding necrotic or cauterized tissue.
When all of the tissue has been trimmed, add 5 to 7 milliliters of 37 degrees Celsius liquid agarose into a 2-cubed centimeter plastic embedding mold, and chill the agarose on a bed of ice for one minute. Place 2 to 4 strips of tissue into the agarose, with the long axis oriented vertically, leaving the tissues on ice for another 2 to 5 minutes after their placement.
When 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 advanced 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.
