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

Animal procedures described here were performed with the approval of the Institutional Animal Care and Use Committee (IACUC) at the Department of Laboratory Animal Resources, Roswell Park Comprehensive Cancer Center, Buffalo, New York.

NOTE: Male mice to be dissected to isolate prostates or prostate tumors for generation of organoids should have at least reached the age of sexual maturity — about 8-10 weeks of age. Specific ages of mice can vary amongst studies. Some factors to consider when choosing age include age-dependent changes in prostate cell populations, age-dependent expression of specific promoter-driven Cre transgenes, and rate of prostate tumor progression in a particular GEMM.

NOTE: Figure 1 shows a pictorial description of the procedure for generation of tumor organoids.

1. Preparation

1. Prepare mouse prostate organoid media according to Drost et al. with the following alterations. Use conditioned medium instead of recombinant proteins for Noggin and R-Spondin and use a final concentration of 1% (v/v), instead of 10%, for both Noggin- and R-Spondin-conditioned medium.
   NOTE: HEK293 cells stably transfected with HA-mouse Noggin-Fc or HA-mouse Rspo1-Fc are used to produce Noggin- or R-Spondin- conditioned medium, respectively. These cell lines were a gift from the Calvin Kuo Laboratory at Stanford University.
2. Prepare digestion solution in a 15 mL tube by diluting 20 mg/mL collagenase II with Advanced DMEM/F12(+++) media to a final concentration of 5 mg/mL. Add Y-27632 Rock Inhibitor to the collagenase II solution at a final concentration of 10 µM.
   NOTE: The ratio of digestion buffer to tissue is 1 mL to 50 mg, which we use according to Drost et al.

2. Mincing and Digestion of Tumor Tissue

1. In the cell culture hood, place prostate tumor tissue in a sterile 10 cm culture dish, dissect and discard necrotic tissue.
2. Mince the remaining prostate tumor tissue into 1 mm³ cubes by holding the tissue pieces with the sterile curved forceps and cutting with dissection scissors.
3. Place the minced tumor pieces in the 15 mL tube with the digestion buffer by scooping them up with the curved side of the forceps. Digest the tumor tissue at 37 °C with shaking for 1.5 to 2 h. Check digestion progress every 20 min.
   NOTE: At this time, take out at least 2 mL of matrix from -20 °C storage and thaw on ice. 1 mL aliquots of matrix will take approximately 3 h to thaw.
4. After tissue digestion, centrifuge tube at 175 x g for 5 min at 4 °C to form a cell pellet.
5. Remove the supernatant, flick the tube to loosen the cell pellet, and resuspend the cell pellet in 1 mL of pre-warmed trypsin supplemented with 10 µM Y-27632 Rock Inhibitor. Put the tube in a 37 °C water bath for 5 min.
6. After incubation, pipette up and down 5 times with a standard P1000 tip. Return the tube to 37 °C water bath for another 5 min and repeat step 2.6.
   NOTE: At this time in the procedure, warm a sterile 6-well cell culture dish by putting it into a 55 °C incubator.

3. Counting Cells and Resuspension in Matrix

1. Wash the cells by adding 9 mL of cold AdDMEM/F12(+++) and centrifuge the tube at 175 x g for 5 min at 4 °C.
2. After centrifugation, remove the supernatant, flick the tube to loosen the pellet, and wash the cells again by adding 10 mL of cold AdDMEM/F12(+++). Centrifuge the tube at 175 x g for 5 min at 4 °C.
3. After centrifugation, remove the supernatant, flick the tube to loosen the pellet, resuspend the cells in 1 mL of AdDMEM/F12(+++), and count the number of cells using a hemocytometer according to standard procedure.
4. Seven-to-eight domes fit in one well of a 6-well dish when organoids are plated in matrix via a drop-wise fashion. Approximately 200 μL of matrix will produce 7-8 domes. Decide how many wells of organoids are needed for future experimental purposes and calculate the volume of matrix required. Then, calculate the volume of cell-containing solution needed to have a final concentration of 1.0 x 10⁶ cells/mL of matrix.
5. After counting cells, centrifuge at 175 x g for 5 min at 4 °C. Remove the supernatant, flick the tube to loosen the pellet, and resuspend the cells in volume of matrix calculated in step 3.4.
   NOTE: Matrix remains in liquid form only at 4 °C; keep matrix stock tubes and matrix-cell solutions on ice at all times.

4. Plating Matrix Domes and Application of Media

1. Mix matrix-cell solution with a P200 pipet to evenly distribute the cells without introducing bubbles. Remove the 6-well culture dish from the 55 °C incubator.
2. Carefully pipette 200 μL of matrix-cell solution and quickly drop the solution into a well to create domes.
3. Repeat step 4.2. until volume of matrix-cell solution is spent. Allow the domes to solidify at room temperature for 2 min.
4. Flip the 6-well dish upside-down and put the dish into a 37 °C incubator to continue solidification for 20 min.
5. After incubation, add 2 mL of mouse prostate organoid media to each well. Add synthetic androgen R1881 to each well for a final concentration of 1 nM and Y-27632 Rock Inhibitor to a final concentration of 10 μM. Mix carefully and place the plate in a 37 °C incubator for culturing.
   NOTE: Organoid culture media needs to be supplemented with 10 µM Y-27632 Rock Inhibitor for only 1 week after organoid generation.

Figure 1: Flow chart of the protocol for generating prostate tumor organoids. After dissecting the prostate tumor, mince the tissue into 1 mm pieces. Digest the tumor pieces in collagenase, collect the cells, and digest in trypsin to obtain a single cell suspension. After counting cells, resuspend in volume of matrix required for a 1.0 x 106 cell/mL cell concentration. Plate domes in dish using a drop-wise method. Please click here to view a larger version of this figure.