Preparing Organoids from Tumors: A 3D In Vitro Tumor Model

Mince the tumor tissue into small pieces. Transfer the pieces into a tube with digestion buffer. Collagen fibers surrounding the cells get broken down, degrading the tissue's matrix. Then, pellet the digested pieces and discard most of the digestion buffer. Flick the tube to break up the pellet.

The solution now consists of various-sized cell clumps. Resuspend the cell clumps in a solution of trypsin and a ROCK inhibitor. The trypsin continues breaking down the tumor and the inhibitor prevents cell death. At regular intervals, pipette the solution to break up the clumps and disperse the cells. Then, resuspended the cells in matrix solution at the appropriate concentration.

Place drops of the solution in a culture plate to form matrix domes. Place the plates upside down in an incubator and allow the matrix to completely solidify. Then, add cell culture media and incubate, right side up. The matrix provides a scaffold to support three-dimensional growth of the organoid.

After mincing, place the tumor pieces into a 15-milliliter tube of digestion buffer for 1.5 to 2 hour digestion at 37 degrees Celsius. At the end of the digestion, sediment the digested tissue by centrifugation and discard the supernatant.

Flick the tube to loosen the cell pellet. Resuspend the cells in 1 milliliter of pre-warmed trypsin supplemented with 10 micromolar Y-27632 ROCK inhibitor for 5 minutes at 37 degrees Celsius.

At the end of the incubation, triturate the tissue slurry five times with the standard P1000 pipette tip. Return the tube to the water bath for an additional 5-minute incubation and trituration.

For matrix dome plating, wash the digested cells in 9 milliliters of cold medium, then collect the cells by centrifugation. Resuspend the cells in 1 milliliter of fresh medium for counting, and after counting, collect the cells by centrifugation once again. Dilute the tumor cells in the appropriate volume of matrix.

Carefully drop 200 microliters of matrix cell solution into each well of a 55 degree Celsius warmed 6-well plate. Allow the domes to solidify at room temperature for 2 minutes before placing the plate upside down in a 37 degrees Celsius incubator for 20 minutes.

When the domes have fully solidified, add 2 milliliters of prostate organoid medium supplemented with androgen R1881 and ROCK inhibitor to each well. Return the plate to the cell culture incubator.

• In vitro tumor models - Methods for studying tumor behavior in lab-controlled conditions.
• PeproTech Noggin - A protein used to regulate cellular processes in tissue culture.
• Tumor model - A method or process used to replicate tumor environment and behavior for scientific studies.
• Cell pellet - Cluster of cells collected at the bottom of a tube after centrifugation.
• ROCK inhibitor organoids - Compounds that prevent cell apoptosis during organoid creation.
• Noggin conditioned media - Media enriched with Noggin protein, used in cellular culture and experiments.
• Cell pellet in tube - Term refers to cells that have been concentrated by centrifugation in a sample tube.

• Introduce In vitro tumor models by defining this as a replicate of tumor behavior (e.g., in vitro tumor models).
• Key Concepts by summarizing principles involved in cell pellet creation (e.g., cell pellet).
• Underlying Mechanisms through visualizing the process of using ROCK inhibitor in organoid creation.
• Connect the process of tumor tissue mincing to the creation of a cell pellet and suspension in a matrix solution.

• What are in vitro tumor models and how to create a cell pellet in lab conditions?
• What role does the PeproTech Noggin play in the tumor model creation?
• How does the ROCK inhibitor assist in the creation of organoids?

• Practical Applications - Tumor models help study cancer growth and potential cures (e.g., in vitro tumor models).
• Industry Impact - Lab studies benefit the healthcare industry and the field of oncology (e.g., ROCK inhibitor organoids).
• Societal Importance - The research contributes to advancements in cancer treatment options (e.g., Noggin conditioned media).
• Link to Scientific Advancements - Understanding tumor behavior facilitates innovative treatment approaches.