CRC Organoid Cell Labeling: A Method to Generate GFP Lentivirus-transduced Colorectal Cancer Organoid Cells
Transkript
– 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 organoid formation. Observe the culture under a fluorescence microscope. Transduced cells synthesize green fluorescent proteins. Organoids formed from these cells display GFP positivity. The following protocol demonstrates GFP lentiviral transduction into patient-derived colorectal cancer organoids cells.
– After 7 to 10 days of culture, detach the organoids mechanically with a sterile cell scraper and transfer the organoids into 1.5 milliliter microcentrifuge tubes for their centrifugation. Then, resuspend the pellets in 500 microliters of PBS with gentle tapping for another centrifugation. To dissociate the colorectal cancer organoids, add 500 microliters of proteolytic and collagenolytic enzyme solution to the tubes and mix with gentle tapping.
After 10 minutes at room temperature, add 500 microliters of medium supplemented with 1% FBS to the cells, and very gently pipette the cell suspension several times to break up the organoids.
– Gentle pipetting of human CRC organoids during passaging is critical to maintaining an intact spheroid formation in culture.
– Centrifuge the cells again and resuspend the pellets in 100 microliters of 5x GFP lentivirus stock and 400 microliters of fresh colorectal cancer organoid culture medium.
– It is essential to infect the human CRC organoids with a high titer of GFP lentivirus, particularly to achieve a nearly 100% infection efficiency.
– Adjust the volume in each tube to achieve a 5 times 10 to the fifth dissociated tumor cell per 500 microliters of medium concentration, and plate 500 microliters of cells into each well of an artificial extracellular matrix coated 12-well plate as demonstrated. After 18 hours in the cell culture incubator, transfer the floating cell-containing supernatants into individual 1.5 milliliter microcentrifuge tubes for centrifugation, followed by resuspension of the pellets in 70 microliters of artificial extracellular matrix per tube on ice.
To enhance the colorectal cancer cell viability, overlay the tumor cell-containing artificial extracellular matrix onto the tumor organoids in the 12-well plate and place the plate in the cell culture incubator for 30 minutes. When the artificial extracellular matrix coating has solidified, feed the cultures with 1 milliliter of fresh CRC organoid culture medium supplemented with 1% FCS and return the cells to the cell culture incubator for three days. Then, observe the infected cells by fluorescence microscopy to confirm a nearly 100% GFP positivity and return the cells to the incubator for another 4 to 6 days.
