Tumor Enzymatic Digestion: A Procedure to Generate Tumor Cell Suspension from Patient-derived Xenograft Cancer Mouse Models

- Patient-derived xenograft, or PDX, models are experimental animal models bearing tumor cells or tumor fragments implanted directly from a human patient. These models simulate the human tumor tissue microenvironment, allowing tumor propagation. To generate tumor cell suspensions, first, harvest the patient-derived tumor portion from the recipient mouse.

Mince the tumor segments to obtain smaller fragments. Treat the fragments with a digest solution containing a cocktail of collagenase, hyaluronidase, and deoxyribonuclease enzymes. Collagenase and hyaluronidase enzymes digest the extracellular matrix that holds the tissue together, facilitating the release of individual cells into the suspension.

The deoxyribonuclease degrades any free DNA released from lysed cells. Now, filter the digested sample through cell strainers to remove any undigested tissue and debris and obtain single-cell suspensions. Centrifuge to separate the cells. Discard the supernatant containing the digestion mix and resuspend the cell pellet in fresh media.

Finally, supplement with lymph node stromal cells in the desired ratio to enhance the metastatic potential of the tumor cells. In the following protocol, we will show an enzymatic digestion-based generation of luciferase-labeled tumor cell suspension from patient-derived xenograft mouse models of colorectal cancer.

- After removing patient-derived tumors from euthanized mice, use sterile surgical scissors to mince luciferase-positive tumor pieces as small as possible in a Petri dish under a laminar flow hood. Transfer the tumor pieces into a sterile 50 milliliter conical tube.

To prepare digest solution, add 10 milliliters of collagenase type IV, 80 microliters of hyaluronidase, and 160 microliters of deoxyribonuclease type I to 40 milliliters of HBSS, and mix by inverting. Add 35 to 40 milliliters of the digest solution to minced tumor and incubate at 37 degrees Celsius with continuous rotation for two hours with periodic vigorous shaking.

To remove debris after digestion, filter the digested tumor through sterile 100 micrometer cell strainer, followed by filtration through a 40 micrometer cell strainer, saving the flow-through in a 50 milliliter tube each time and discarding the debris. To wash free cells, add 20 milliliters of HBSS to the flow-through and centrifuge at 329 x g for 5 minutes.

Aspirate the supernatant and resuspend the pellet in 30 milliliters of HBSS. Transfer 100,000 tumor cells to a sterile 15 milliliter conical tube and add 300,000 previously prepared, fresh HK cells. Centrifuge the cells at 329 x g for 5 minutes and discard the supernatant by aspiration. Resuspend the cells in complete RPMI medium and keep on ice until ready for use.