Spheroids are in vitro cultures of cells self-aggregated to form three-dimensional spherical structures. For propagating tumor spheroids, begin with spheroids cultured in a suitable extracellular matrix, or ECM, contained in a culture plate.

Triturate using a micropipette to break the ECM mechanically and loosen the spheroids. Transfer the spheroid-ECM mix to a microcentrifuge tube.

Centrifuge at a low speed to pelletize the spheroids and separate them from the ECM in the supernatant. Discard the spent ECM-containing supernatant. Transfer the tube containing the spheroid pellet on ice to prevent ECM solidification during subsequent steps.

Supplement the pellet with fresh liquefied ECM. For sub-culturing, mix the contents by pipetting up and down repeatedly to dissociate the spheroid cells and release them into suspension.

Seed this mixture containing spheroid-forming cells and ECM into a fresh culture dish. ECM solidifies, enabling the entrapment of tumor cells. Add the desired growth medium. Incubate to facilitate the growth of entrapped cells into new spheroids.

For shipping purposes, transfer the spheroid-ECM mixture into a culture flask. Allow the ECM to solidify and entrap the spheroids within.

Fill the flask with growth media to avoid cell-shearing and to maintain the viability of the spheroids. Transfer the flask to a shipping package.

Mechanically break the ECM with a P1000 pipette and transfer it to a sterile 1.5-millimeter tube. Centrifuge the tube at 1,000 x g at 4 degrees Celsius. Then, remove all supernatant and place the tube on ice. Add two to four times the volume of new ECM to the pellet and mix the contents of the tube by pipetting up and down 10 times, making sure to avoid introducing air bubbles.

Transfer 5 to 20 microliters of the ECM in SBNET mixture to a new 96-well plate and allow the ECM to solidify. Then, cover the solidified ECM with new SBNET medium and put the plate in the incubator. If shipping SBNET spheroids to another lab, transfer the spheroids with the new ECM to a T25 flask and allow the ECM to solidify. Fill the flask with SBNET spheroid medium, screw the cap on tightly, and prepare the shipping package.

• Mechanical Breaking - Process of physically separating materials such as ECM by pipetting.
• Centrifuge - Device used to separate substances by spinning them at high speed.
• Supernatant - The liquid lying above a solid residue after centrifugation.
• SBNET medium - Special medium used for sustaining spheroids in lab conditions.
• Spheroids - 3D cell cultures used in scientific experiments.

• Introduce Mechanical Breaking - This method involves physically separating ECM (e.g., pipetting).
• Outline Centrifuge process- Spinning at high speed to separate substances (e.g., sedimentation).
• Explain Supernatant- Liquid that stays above solid residue post centrifugation.
• Connect to SBNET medium - Essential medium to sustain spheroids during the experiment.

• How is the process of mechanically breaking the ECM conducted?
• What is the purpose of using a centrifuge in the experiment?
• What is supernatant and its role in the experiment?

• Practical Applications - Real-world use cases of these techniques (e.g., research labs).
• Industry Impact - Various scientific sectors benefitting (e.g., biotechnology).
• Societal Importance - Possible healthcare advancements (e.g., drug discovery).
• Link to Scientific Advancements - Innovations enabled by these techniques.