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.