The overall goal of this procedure is to generate stably transduced organoids from primary mouse intestinal epithelium for downstream analysis of intestinal organoids. This is accomplished by first transecting HEC 2 93 T cells with lentiviral packaging vectors and lentiviral plasmid, encoding the gene of interest to produce high titer lentiviral particles. The second step is to pretreat cultured mouse intestinal organoids with a number of growth factors to obtain cystic hyper proliferative crypts.
Next, transduce the organoids with the high titer lentivirus and incubate the transduced organoids in matrigel. The final step is to embed full-grown transduced organoids in paraffin for downstream immunohistochemistry analysis. Ultimately, lentiviral organoid transduction is used to generate genetic alterations in an in vitro model of the intestinal epithelium that is reliable, reproducible and fast.
The main advantage of this technique over existing methods such as cancer cell lines, is that organoids are non-mutated displaced stem cell hierarchy. In text cellular polarization, it can differentiate into all different cell types of the small intestinal epithelium. In addition, transduced organoids can be used to study specific genetic elements, thereby outweighing the use of transgenic mice and facets of cost and speed.
The production of lentiviral particles is a five day protocol that begins with splitting HEC 2 93 T cells to 60 to 80%co fluency in a 162 square centimeter flask in cell culture medium. Incubate the cells overnight in a humidified cell culture incubator at 37 degrees Celsius on the following day. Prepare the DNA transfection solution and the polyethylene amine or PEI transfection solution as described in the protocol text.
Add the DNA transfection solution to the PEI solution vortex or invert a number of times and incubate for five minutes. At room temperature. Drip two milliliters of the DNA TRANSFECTION plus PEI solution onto the HEC 2 93 T cells and incubate for four hours at 37 degrees Celsius after four hours.
Refresh the culture medium. To remove the PEI incubate the cells for two days at 37 degrees Celsius on day four, collect the supernatant in a 50 milliliter tube. Add new culture medium to the cells and return the flask to the incubator centrifuge the collected supernatant at 500 Gs for five minutes.
To remove dead cells, then use a large 60 milliliter syringe to push the SUPERNAT through a 0.45 micrometer filter. Store the filtered supernat overnight at four degrees Celsius on the following day. Collect centrifuge and filter the second batch of supernatant as shown for the first batch.
Pool the two batches of supernatant in ultra centrifuge tubes. Centrifuge at 50, 000 GS for 90 minutes. When the centrifugation is complete, very carefully, remove the capsules containing the ultra centrifuge tubes and put them into a laminar flow hood.
Open the capsule holding the ultra centrifuge tube and decant the medium carefully with the pellet on the upper side of the tube. It is important to remember which side of the tube appellate would've formed since viral pellets may be difficult to visualize. Next, use a micro pipette to remove the last bit of medium while taking care not to agitate the opaque brown pellet that is visible on the side of the bottom of the ultracentrifuge tube.
Re suspend this pellet in 500 microliters of organoid culture medium supplemented with nicotinamide kinase inhibitors and poly brain resus. Suspension in this medium is important since high titer virus will be used directly for transduction of organoids two days prior to transduction. Split a full 0.95 square centimeter well of organoids into a new well of a 48 well plate.
Following a previously published protocol. Aim to obtain approximately 50 small organoids in organoid culture. Medium supplemented with a glycogen synthase kinase inhibitor and nicotinamide.
To obtain cystic hyper proliferative crypts. Incubate the organoids for two days on the day of transduction harvest organoids with a P 1000 micro pipette. Pipette the matra gel and medium up and down, thereby disrupting the mixture.
Place the mixture in a 15 milliliter tube. Disrupt the mixture further using a PEs your pipette in which the distal opening has been decreased by melting Pellet the organoids by centrifuging at 100 Gs for five minutes. Then remove the supernatant at 500 microliters of prewarm one x trypsin and resuspend the organoids incubate for three minutes.
In a 37 degree Celsius water bath, inactivate the trypsin by adding 3.5 milliliters of cell line culture medium centrifuge for five minutes. At 500 Gs.Remove the super natan to leave the pellet in approximately 20 microliters of medium. Transfer the organoids in this last small amount of medium into a well.
On a 48 well plate. Add the previously prepared high titer lentivirus in transduction medium to the organoids and resuspend. Incubate the organoid virus mixture for one hour at 37 degrees Celsius in a culture incubator to allow transduction after one hour, add one milliliter of organoid culture, medium to the organoid virus mixture, resuspend, and transfer the mixture to a micro centrifuge tube centrifuge at 850 GS for five minutes.
To pellet the organoids, remove the supernatant and resuspend the pellet. In 20 microliters of ice cold matra gel, put the droplet in the middle of a well. In a 48 well plate and incubate at 37 degrees Celsius for 15 minutes.
For the droplet to solidify after 15 minutes. Carefully add 250 microliters of organoid culture. Medium supplemented with nicotinamide and kinase inhibitors.
Prewarm an aluminum block in an incubator at 70 degrees Celsius to keep the paraffin liquid. Remove the medium from a single well with full-grown organoids leaving the embedded organoids intact and add one milliliter of 4%Paraform aldehyde in PBS directly to the well. Replace the paraform aldehyde with one milliliter of PBS.
Resuspend the fixed organoids in the PBS and transfer to a glass vial. Let the organoids sink to the bottom for one minute. Next, pipette off the PBS and replace with 70%ethanol in which a couple of droplets of eoin solution are dissolved.
To enable visualization of organoids throughout the embedding process, leave the organoids in 70%ethanol at room temperature for 30 minutes. Remove the 70%ethanol by carefully pipetting. The organoids can be visualized by eye because of the pinkish eoin color.
Replace the embedding solution with 96%ethanol and leave at room temperature for 30 minutes in this manner. Subsequently, pass the organoids through 70%ethanol, 90%ethanol, 96%ethanol, 100%ethanol, 100%ethanol, xylene and xylene. Decant the last xylene wash and pour paraffin into the glass vial.
Put the vial immediately in the prewarm aluminum block at 70 degrees Celsius for 30 minutes. After 30 minutes, replace the paraffin with new clean paraffin with a prewarm, PE or pipette. Pour the paraffin off and use a prewarm pest or pipette with a large opening to pipette the organoids into the paraffin block mold into a layer of liquid paraffin.
Warm a dissection needle with bunsen burner and manipulate all organoids as much as possible toward the center of the paraffin block mold. When localization of the organoids in the mold is satisfactory, chill the mold slightly to solidify the paraffin layer. Finish the block by pouring more paraffin on top and add a standard histological embedding cassette.
In this protocol, organoids of intestinal epithelium were transduced with lentiviruses. Normal organoids grow as K crypt villous structures with a number of crypts that come out of a shared villus domain. Upon treatment of these organoids with the GSK three B inhibitor, CHIR 9 9 0 21, proliferation increases and crypts become cystic after organoid transduction.
Using lentiviral EGFP overexpression, the organoids express fluorescent EGFP transduction enhancement steps that may be performed when transduction efficacy is low, such as inoculation or prolonged viral incubation are not required, as these steps will not increase the already high efficacy. Subsequently, these organoids are processed for RNA techniques and immunohistochemistry. This representative image is a section of a mouse intestinal organoid, stained for BRDU after incubation with BRDU for one hour prior to fixing.
After watching this video, you should have a good understanding of how to transduce organoids from primary intestinal epithelium using lentiviral or retroviral particles.
