Because organoids recapitulate the native structural organization, as well as many molecular features of intestinal epithelium in vivo, this technique allows us to study normal biology and disease states in the laboratory. The main advantage of this technique is the ability to genetically engineer organoids using a very highly efficient transduction approach with low cytotoxicity, thus allowing for functional studies of these genetically manipulated organoids. The implications of this technique extend toward therapy for intestinal disease because we can expose intestinal organoids to drugs and other agents or interventions.
This method can be applied to study developmental and pathologic processes in other systems amenable to organoid cultures, such as breast tissues or for cells growing under standard 2D conditions. Generally, individuals new to this method will succeed with transduction in difficult-to-engineer organoids or other cells. We first had the idea for this method when we encountered difficulties transducing our organoids and stem cells.
Visual demonstration of this method is critical as structural loss of the organoids can occur if samples are not properly treated during the embedding process into the basement matrix for cryosectioning. To grow organoids for viral transduction, seed organoid cell cultures with 200 microliters of transduction medium per well in a 48-well plate, and incubate in a standard tissue culture incubator. Thaw vials of virus for transduction, allowing for about 50 microliters of concentrated virus for the transduction of each well in 48-well plates.
In a 1.5-milliliter tube, mix the virus with 12 microliters of a magnetic nanoparticle solution, and incubate at room temperature for 15 minutes. After 15 minutes, add the magnetic nanoparticle solution and virus mixture to the cells to be transduced. Place the 48-well plate on a magnetic plate, and incubate in a standard tissue culture incubator for at least two hours.
When the viral transfection is complete, transfer the infected organoid cell clusters and transduction medium from each well into a 1.5-milliliter tube. Centrifuge at 500 times g for five minutes. Discard the supernatant with gentle suction, and cool the tubes containing the pellet on ice for five minutes.
Add 120 microliters of basement membrane matrix to each tube, and resuspend the pellet by slowly pipetting up and down. Seed 30-microliter drops of the matrix-cell mixture into a new 48-well plate. Incubate the plate at 37 degrees Celsius for five to 15 minutes until the matrix solidifies.
Add transduction medium to each well, and incubate in a standard tissue culture incubator for three to four days. After three to four days, inspect the cultures under a light microscope at 10x magnification to ensure organization of cell clusters into organoid structures. Gently replace the transduction medium in each well with 250 microliters of organoid culture ENR medium.
Return the plate to the incubator. Begin this procedure by removing the ENR medium by gentle suction, being careful not to perturb the basement membrane matrix. Gently wash with 500 microliters of PBS.
Fix the organoids by adding one microliter of 4%paraformaldehyde solution per well, and incubate at room temperature for 30 minutes. After 30 minutes, remove the paraformaldehyde solution by suction. Gently add one microliter of PBS per well, and then remove the PBS by gentle suction.
In this manner, wash twice with PBS. Remove the PBS from the second wash, and add one microliter of 30%sucrose buffer to each sample. Incubate the fixed organoids in sucrose for one hour at four degrees Celsius to dehydrate the samples.
Remove the sucrose buffer by suction, and add just enough embedding compound to cover the matrix layer in each well. Incubate at room temperature for five minutes. Next, place the samples in a minus 80-degree Celsius freezer for 10 minutes or until the embedding compound turns solid and white.
Place the plate with frozen embedding compound at room temperature to allow for minimal melting of the compound along the edges. Use a scalpel or spatula to separate the block from the walls of the well. Work quickly to prevent melting, using forceps to remove the matrix embedding compound block, and place it in a specimen block.
Fill the mold completely with embedding compound. Freeze at minus 80 degrees Celsius for 30 minutes before using the block for sectioning. This protocol was optimized by first transducing intestinal organoids with lentiviral vectors linked to GFP.
The GFP was visualized at each stage in organoid development, including early on when crypt cells organize into cyst-like structures or at later time points when organoids form buds. Successfully transduced intestinal organoids were then formalin-fixed, cryosectioned, and analyzed by immunofluorescence imaging. In this image, nuclei are stained in blue, and the green indicates epithelial cell adhesion molecules, which demarcates cell borders.
The red indicates lysosome staining to identify Paneth cells. Once mastered, transducing organoids can be performed in about three hours, and organoids crysection embedding can be done in about 2 1/2 hours. When attempting this procedure, it is important to remember to work on ice when handling basement matrix reagents.
After completing this procedure, immunofluorescent analysis and other methods to detect proteins or organelles can be performed to assess localization or relative abundance. This technique paves the ways for researchers to explore normal biology and disease states in vitro and also for in vivo studies if the transduced organoids or cells can be implanted into mice. After watching this video, you should have a good understanding of how to use magnetically labeled viruses to genetically engineer organoids for cryosectioning.
Please don't forget that working with viruses can be extremely hazardous, and precautions such as wearing appropriate protective gear should always be undertaken when performing this procedure.
