CRC Organoid Cell Labeling: A Method to Generate GFP Lentivirus-transduced Colorectal Cancer Organoid Cells

- Organoids are three-dimensional in-vitro cellular constructs that self-organize to mimic key tissue characteristics of its organ of origin. To begin labeling, treat harvested organoids with a proteolytic and collagenolytic enzyme cocktail to dissociate the cells into suspension. Centrifuge to separate the cells from enzyme-containing supernatant. Discard the supernatant. Dissolve the cell pellet using a medium containing lentiviral vectors. These recombinant viruses carry the coding sequence for a green fluorescent protein or GFP.

The viruses fuse with the cells and release their genetic material and certain viral enzymes into the cytoplasm. The viral reverse transcriptase synthesizes the complementary viral DNA that moves into the host's nucleus. The viral integrase helps incorporate the GFP sequence-containing viral DNA into the host genome. Grow these transduced cells in an extracellular matrix or ECM-coated culture plate. Aspirate the medium and centrifuge to collect any unattached cells. Resuspend the cells in additional ECM.

Overlay the suspension on the adherent culture to promote cell viability. Once the matrix solidifies, add culture medium and incubate to facilitate org