A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

The overall goal of this procedure is to demonstrate how to perform functional genetic studies in organoids. This is accomplished by first pre-treating the organoids with WINT three A containing media to facilitate their adoption of a cystic shape and to increase their stem cell numbers. In the second step, the virus is produced and the organoid fragments are then transduced and inoculated.

In the final step, the transfected organoids are seated and then selected for stable viral integration. Ultimately, the expression or suppression of the gene of interest can be assessed by GFP fluorescence. The main advantage of using this technique over existing AL models like the mouse, is that with this technique, functional genetic experiments can be performed in vitro with the intestinal organoids.

Demonstrating the procedure will be Amanda Andro, a PhD student from my laboratory To isolate the crypts, begin by washing the small intestine with pre chilled calcium and magnesium free PBS. Then cut the tissue into three to five centimeter long pieces and open the pieces longitudinally with scissors. Use forceps to spread the tissues open, scrape off excess villi using a cover slip, and then transfer the pieces to a 50 milliliter tube containing chilled PBS.

Wash the tissue fragments two to three times with vigorous shaking, changing the PBS until the PBS turns less cloudy after the last wash, incubate the tissue in a 50 milliliter tube containing 30 milliliters of PBS supplemented with one millimolar EDTA at four degrees Celsius on a tube roller. After 30 minutes vigorously, shake the tube again and transfer the tissue to another 50 milliliter tube containing 30 milliliters of PBS supplemented with five millimolar EDTA. After an hour incubation at four degrees Celsius on the tube roller vigorously.

Shake the tube again and transfer the solution to a new 50 milliliter tube. Then count the number of crypts in 50 microliters. Calculate and transfer the volume containing 50 to 100 crypts into a 1.5 milliliter tube.

Next, spin down the crypts and resuspend the pellet in 50 microliters of basement matrix. Then seed the crypt solution into a Prewarm 24 Well plate and incubate the plate in a tissue culture incubator for five to 15 minutes. Once the basement membrane has polymerized, incubate the matrix with 500 microliters of ENR medium in a cell culture incubator.

Refreshing the media every three days approximately 24 hours after seating, the organoids will exhibit a small round cystic shape. After another two to three days, budding structures will become visible while the organoids are adopting their cystic morphology Seed, approximately five times 10 to the six platinum E cells in a 150 millimeter dish with 15 to 18 milliliters of media supplemented with pur, mycin and blastin. After two to three days, change the medium to media without antibiotics and add 30 micrograms of retroviral DNA construct and 240 microliters of pi to two individual tubes each containing one milliliter of optimum.

Next, mix the tubes and incubate them at room temperature. After five minutes, pool the two solutions and incubate them at room temperature for another 20 to 30 minutes. Add the complete mixture to the platinum E cells and carefully shake the plate to ensure equal distribution of the DNA PI complexes.

After an overnight incubation, refresh the medium and incubate the cells again. After two days, collect only the medium in a 50 milliliter Falcon tube and strain it through a 0.45 micron filter. Then centrifuge the solution for 12 to 16 hours at 8, 000 times G and four degrees Celsius, and resuspend the pellet in 250 microliters of transduction medium.

To prepare the organoid fragments for retroviral transduction, first pipette the culture 30 to 50 times with a 200 microliter pipette tip to mechanically disrupt the organoids, the solution will become cloudy and no whole organoids should be detected. After spinning down the fragments, incubate the pellet in 500 microliters of cell culture, grade recombinant protease at 37 degrees Celsius for five minutes. Following the incubation, use a light microscope to assess the size of the organoid fragments and to count the number of cells per fragment.

Then add 500 microliters of ENR medium to terminate the dissociation process and spin down the cell fragments. After discarding the supernatant, place the pellet on ice and then seed the fragments into one well of a 48. Well plate in 250 microliters of the previously prepared retroviral solution.

Mix the cell solution gently with a one milliliter pipette tip, and then seal the plate with param to perform the inoculation first centrifuge. The 48 well plate for one hour at 600 times G and 32 degrees Celsius. Then carefully remove the para film and incubate the plate for six more hours in a cell culture incubator.

After the incubation, transfer the infected fragments to a 1.5 milliliter tube. Then spin down the organoid fragments again and chill the pellet on ice for five minutes. Once the pellet has cooled, add 100 microliters of basement matrix to the tube and slowly pipette the pellet up and down to resuspend it.

Then seed 50 microliter drops of the fragment matrix suspension into each well of a 24 well plate and incubate the plate at 37 degrees Celsius for five to 15 minutes After the basement matrix has solidified, incubate the organoids with transduction media without poly brain. After two to three days, add pur mycin containing media to the wells to start the selection. When the fragments start to form organoids, replace the transduction media with ENR media supplemented.

With puram Mycin organoids are ready to be split when their central lumens become darkened due to the presence of dead cells. After two to three days of Wint three a media pre-treatment, the organoids should adopt around cystic morphology, increasing the number of stem cells, and enhancing the chances of obtaining a stable integration in the stem cells. In these images, the fluorescent protein expression from the M-S-C-V-E-G-F-P retrovirus can be observed in the cells originating from the surviving organoids that acquired the expected cystic morphology within two to three days of transduction.

While attempting this procedure, it's important to check the size of the organoid fragments as it strongly affects their survivability. After watching this video, you should have a good understanding of how to perform retroviral transduction of organoids.