Source: Okazawa, Y. et al. High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor. J. Vis. Exp., (2018).
This video describes the detailed protocol for generating 3D organoids from murine-model derived colorectal cancer cells by propagating them on artificial extracellular matrix coated plates. These CRC organoids can be used as a model to study the primary growth of human tumor cells and metastasis.
All procedures involving animals have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Extraction of PDXs into the Cell Suspension for the CRC Organoid Culture
Experimental procedures for dissociation of CRC PDXs are outlined in Figure 1B (step 3).
2. Generation of the CRC Organoids Cultured on Artificial Extracellular Matrix
Experimental procedures for the CRC organoid culture of the colon PDXs are outlined in Figure 1B (step 4).
Figure 1: Schematic representation of generation of metastases by the PDX-derived CRC organoids labeled with GFP lentivirus in NOG mice. (A) Implantation of small pieces of the CRC tissue subcutaneously into NOG mice (step 1). The CRC tissue surgically dissected from the patient was cut into pieces and implanted subcutaneously into NOG mice. s.c.: subcutaneous implantation. (B) Generation of CRC organoids dissociated from PDXs (step 2–4). The developed CRC xenografts were minced (step 2) and transferred into a 15 mL tube containing the culture medium including collagenase (step 3). After incubation with slow agitation, the CRC cell suspension was filtered (step 3). Then, the organoid cell suspension in the CRC organoid medium was seeded onto an artificial extracellular matrix-coated plate and incubated overnight in a CO2 incubator (step 4). The CRC organoid cells attached to the artificial extracellular matrix were also coated with an additional artificial extracellular matrix and incubated in a CO2 incubator (step 4). s.c.: subcutaneous implantation. (C) Generation of CRC organoids transduced by GFP lentiviral particles prior to employing injection into recipient mice (step 5–7). The GFP lentiviral particles were generated at a high titer (step 5). The PDX-derived CRC organoids grown on an artificial extracellular matrix were directly harvested with a cell scraper and transferred into a microtube (step 6). After centrifugation, the cell pellet was resuspended in PBS. The cell suspension was centrifuged and the cell pellet was dissociated. Then, the CRC organoid cell suspension was incubated with GFP virus stock in the CRC organoid culture medium on the artificial extracellular matrix-coated plate overnight in a CO₂ incubator (step 6). The CRC organoid cells attached to the artificial extracellular matrix were coated with an additional artificial extracellular matrix and incubated in a CO₂ incubator to solidify the artificial extracellular matrix coating (step 6). The CRC organoids were then cultured for periods of 7–10 days to expand cell growth (step 6). To develop a spontaneous metastasis model, the dissociated 5 x 105 CRC organoid cells labeled with GFP suspended in 50 µL of PBS with 50% artificial extracellular matrix were injected orthotopically into NOG mice (step 7). To generate an experimental metastasis model, 4 x 104 CRC organoid cells labeled with GFP in 50 µL of PBS were injected intrasplenically into NOG mice (step 7). o.t.: orthotopic injection, i.s.: intrasplenic injection.
The authors have nothing to disclose.
NOD/Shi-scid IL2Rγ null (NOG) mice | The Central Institute for Experimental Animals,Kanagawa, Japan | Breed 6-week-old male mice under germ-free and specific pathogen free conditions | |
CRC organoid culture medium with 1% or 5% FCS | DMEM/F-12 with GlutaMAXTM supplement (Gibco #10565018) supplemented with 1% or 5% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 ng/ml hEGF and 10 µM Y27632, a ROCK inhibitor. Store at 4°C. Use within 1 month. | ||
Matrigel basement membrane matrix | Corning | #354234 | Store aliquots at -20°C. Place on ice until use |
Collagenase type 1 | Sigma | #C1030 | 150 mg/ml collagenase type1 in 1×PBS. Store aliquots at -20°C for up to 1 year |
Hemocytometer | Erma | #03-202-1 | |
DMEM/F-12 with GlutaMAXTM | Gibco | #10565018 | Store at 4°C. Warm at 37°C before use |
50ml conical tube | Sumitomo Bakelite | MS-57500 | |
microtube | Eppendorf | #0030120086 | Autoclave before use |
40μm cell strainer | Corning | #352340 |