Encyclopedia of Experiments: Cancer Research
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- First, thaw vials of basement membrane matrix at 4 degrees Celsius overnight. The basement membrane matrix consists of proteins present in the tumor microenvironment that help maintain cancer cells' characteristics in vitro. The next day, place the basement membrane matrix vial on ice to retain the matrix in liquid state. Take lung cancer cells into a pre-cooled centrifuge tube and centrifuge at 4 degrees Celsius. Aspirate the supernatant and gently tap to dislodge the pellet and place it on ice. Next, add an appropriate volume of basement membrane mix into this tube and mix to make a uniform cell suspension. Perform all steps at low temperature to prevent the basement membrane from gelling.
Transfer an appropriate volume of cell matrix suspension to the center of each well in a precooled well plate, covering the entire well. Incubate the well plate for the matrix to solidify. Examine under a microscope to ensure even distribution of cells within the matrix. Add 3D culture complete medium into each well and incubate at 37 degrees Celsius. This medium facilitates cell matrix interactions. Thus, the cells form organoids with acinar-like structures. In the following protocol, we will perform the 3D culture of TUM622 lung carcinoma cells.
To begin, thaw vials of basement membrane matrix in a 4 degree Celsius refrigerator overnight. Cool down two milliliter plastic pipettes and tips at minus 20 degrees Celsius overnight. The next day, warm the reagents used to dissociate the TUM622 cells, HEPES buffer, Trypsin-EDTA, and Trypsin Neutralization buffer in a 37 degrees Celsius water bath. Take the thawed basement membrane matrix out of the refrigerator and put the vial on ice. Cool down the tissue culture plates on a metal platform cooler placed on ice. Place centrifuge tubes on a metal cooling rack on ice. Calculate the amount of cells needed, based on the number of wells and the concentration of cells in each well, to be prepared. Transfer TUM622 cell suspension into a cooled centrifuge tube and spin down at 300 times g in a hanging bucket centrifuge at 4 degrees Celsius for five minutes.
With an aspirating pipette attached to an unfiltered tip, aspirate the supernatant carefully, leaving approximately 100 microliters of the medium in the tube. Gently tap on the side of the tube to dislodge and associate the pellet, and then return it to the cooling rack. Using the two milliliter precooled pipettes, gently mix the matrix on ice by pipetting up and down a few times. Pipette at an even and moderate speed so that no bubbles are introduced into the matrix during this procedure.
Transfer 1.1 milliliters of the matrix into each centrifuge tube. Using precooled tips, pipette the matrix in each tube up and down about 10 times to make a uniform cell suspension. Transfer 310 microliters of the cell-matrix suspension into each well of a precooled 24-well plate. Place the pipette at a 90 degree angle to the plate surface and add the suspension to the center of the well. The suspension spreads and covers the entire well. To facilitate downstream immunofluorescence analysis, transfer 60 microliters of cell-matrix suspension into the well center of a 2-well chamber slide. This allows the matrix to form a dome-like structure with much smaller volume.
Return the plate and the chamber slide back into the tissue culture incubator and incubate for 30 minutes to allow the matrix to solidify. After that, examine the plate and slide under a light microscope to ensure that single cells are evenly distributed within the matrix. Add 1 milliliter of prewarmed 3D culture complete medium into each well of the plate and 1.5 milliliters of 3D culture medium into each well of the chamber slide. Then return them to the incubator.