CRC Organoid Culture: A Method to Obtain 3D Organoids from Colorectal Cancer Cells
Published: April 30, 2023
Abstract
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.
Protocol
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). Place the tumor fragments derived from NOD/Shi-scid IL2Rγ null (NOG) mouse on a 10 cm Petri dish. Mi…
Representative Results
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 implant…
Declarações
The authors have nothing to disclose.
Materials
| 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 |
Citar este artigo
CRC Organoid Culture: A Method to Obtain 3D Organoids from Colorectal Cancer Cells. J. Vis. Exp. (Pending Publication), e20333, doi: (2023).