Generating an Organoid Culture from a Brain Tumor Cell Suspension

To make organoids from a single-cell suspension, add an appropriate amount of laminin-rich extracellular matrix or lrECM into a small centrifuge tube in an ice bucket or cold block.

Prepare a cell mixture containing 20,000 cells per organoid. Mix the lrECM cell suspension mixture thoroughly to avoid settling the cells, which would result in non-uniform organoid formation. Next, carefully pipette 20 microliters of the mixture onto a parafilm mold to form a pearl-like droplet. Ensure to cool the pipette tip every two to three organoids to prevent lrECM polymerization.

Once the desired number of organoids are pipetted onto the parafilm mold in a 10-centimeter culture plate, incubate the organoids at 37 degrees Celsius for 1 to 2 hours in a cell culture incubator. After the organoids are solidified, flush the organoids gently off the parafilm mold with NBMC medium using a P1000 tip, and collect them into a new 10-centimeter culture plate containing 20 milliliters of NBMC. Place the culture plate in an incubator without shaking for 4 days.

After 4 days, replace the media in the plate. To do this, place a piece of dark paper beneath the cell culture plate. Tilt the plate and wait for 20 seconds. As the organoids completely settle at the bottom of the dish, slowly remove the media with a large opening glass pipette.

After adding new media, place the plate in the incubator on an orbital shaker at 80 RPM, and exchange media every 2 to 3 days thereafter.