CRC Organoid Cell Labeling: A Method to Generate GFP Lentivirus-transduced Colorectal Cancer Organoid 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 technique of generating CRC organoids transduced by GFP lentiviral particles for enabling fluorescent imaging. These GFP labeled organoids, when placed in animal models, help to study tumor invasion and metastasis.
Protocol
1. 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). Establish a culture medium suitable for the PDX-derived CRC organoids (see Table of Materials, "the CRC organoid culture medium") employing media previously described for human colon organoids. Apply 150 µL of artifi…
Representative Results
Figure 1: Schematic representation of the 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.: subcut…
Divulgaciones
The authors have nothing to disclose.
Materials
| 12-well plate | BMBio | #92412 | |
| microtube | Eppendorf | #0030120086 | Autoclave before use |
| Hemocytometer | Erma | #03-202-1 | |
| 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 |
| DMEM/F-12 with GlutaMAX™ | Gibco | #10565018 | |
| Penicillin | Gibco | #15140122 | Store at 4°C. Use within 1 month |
| hEGF | PEPROTECH | #AF-100-15 | Store at -20°C. Add to medium on same day as use |
| Y27632, a ROCK inhibitor | Wako | #253-00591 | Store at -20°C. Add to medium on same day as use |
| Streptomycin | Gibco | #15140122 | Store at 4°C. Use within 1 month |
| CRC organoid culture medium with 1% or 5% FCS | DMEM/F-12 with GlutaMAX™ 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. | ||
| PRRL-GFP vector | Gift from Dr. Robert A. Weinberg | ||
| the FuGENE 6 transfection regent | Roche | 11814 443001 | |
| pCMV-VSV-G | Gift from Dr. Robert A. Weinberg | ||
| pCMV-dR8.2 dvpr | Gift from Dr. Robert A. Weinberg | ||
| a Zeiss Axioplan 2 stereofluorescence microscope | Zeiss |
Citar este artículo
CRC Organoid Cell Labeling: A Method to Generate GFP Lentivirus-transduced Colorectal Cancer Organoid Cells. J. Vis. Exp. (Pending Publication), e20334, doi: (2023).