Generation and Genetic Manipulation of Human Cervical Organoids

We established a Schumann cervical organoid systems to study cervical biology and pathology, including genetically-engineered organoids to study gene functions and disease mechanisms. This protocol can be applied to study human cervical biology, modern cervical diseases, and screen effective drugs. In addition, this protocol can be modified to establish other epithelial organoid systems.

To begin, place the biopsied human ectocervix tissue sample in a 10 centimeter Petri dish. Use ice cold Dulbecco's PBS without calcium and magnesium to wash the tissue once to remove blood and debris. Transfer the tissue to a 1.5 milliliter tube containing one milliliter of Dispase solution at 2.4 units per milliliter.

Place the tube on a tube rotator set to 20 revolutions per minute and incubate for one hour at 37 degrees Celsius with gentle agitation. Then transfer the sample to a new 10 centimeter Petri dish. Using forceps, carefully separate the white epithelial layer from the rest of the tissue.

Now with sharp forceps, gently peel off the epithelial surface as an intact sheet and immediately transfer the isolated epithelial layer into a 1.5 milliliter tube. Add one milliliter of recombinant trypsin-based dissociation solution to the peeled or scraped sample. Place the 1.5 milliliter conical tube containing cervical epithelium on a tube rotator set to 20 revolutions per minute and incubate at 37 degrees Celsius for five minutes.

Then transfer the cell suspension to a 50 milliliter conical tube and add 10 milliliters of freshly-prepared ice cold blocking buffer composed of PBS with 2%bovine calf serum to stop the enzymatic reaction. Place the tube in a centrifuge and spin at 450 G for five minutes at four degrees Celsius. Discard the supernatant and add one milliliter of red blood cell lysis buffer to the pellet, followed by 20 microliters of deoxyribonuclease and incubate at room temperature for five minutes.

Then wash the cells twice by adding 10 milliliters of ice cold blocking buffer, followed by centrifuging at 450 G for five minutes at four degrees Celsius. After two washes, re-suspend the pellet in 200 to 500 microliters of organoid culture medium. Using a hemocytometer and Trypan blue solution count the cell number.

Re-suspend 500 to 2, 000 viable cells in a final volume of 50 microliters of organoid culture medium in a 1.5 milliliter micro centrifuge tube. Add 50 microliters of basement membrane matrix to the cell suspension and gently mix on ice to avoid bubble formation. Dispense the 100 microliter mixture into the interior of a cell culture insert placed in a 24 well plate.

Place the plate in a 37 degrees Celsius incubator for 20 to 30 minutes until the basement membrane matrix cell mixture solidifies. Next, add 400 microliters of organoid culture medium to the well below the insert after the mixture has solidified. Culture the organoids for one to two weeks until they reach an average diameter of 150 to 250 micrometers.

Add one milliliter of Dispase solution at 2.4 units per milliliter on top of the cell culture insert and gently dissociate the basement membrane matrix. Transfer the Dispase base membrane matrix mixture to a 15 milliliter tube and incubate for one hour at 37 degrees Celsius. Now add 10 milliliters of freshly-prepared ice cold blocking buffer to the cell suspension to stop the enzymatic reaction.

Place the tube in a centrifuge and spin at 450 G for five minutes at four degrees Celsius. Remove the supernatant, re-suspend the pellet in 200 microliters of trypsin and incubate at 37 degrees Celsius for five minutes. Next, add one milliliter of ice cold blocking buffer and gently mix to neutralize the enzyme.

Filter the suspension through a 40 micrometer cell strainer to obtain single cells. Place the tube in a centrifuge and spin at 450 G for five minutes at four degrees Celsius. After discarding the supernatant, re-suspend the cell pellet in 200 to 500 microliters of organoid culture medium.

Using a hemocytometer, and Trypan blue solution, count the cell number. Re-suspend 500 to 2, 000 cells in 50 microliters of organoid culture medium in a micro centrifuge tube and add 50 microliters of basement membrane matrix to the cell suspension. Gently mix to avoid bubble formation and dispense the 100 microliter mixture into the interior of a cell culture insert placed in a 24 well plate.

Place the plate in a 37 degrees Celsius incubator, for 20 to 30 minutes until the basement membrane matrix cell mixture solidifies. Add 400 microliters of organoid culture medium to the well below the insert once the mixture is fully solidified. Then, culture the organoids in organoid culture medium for one to two weeks in a humidified incubator at 37 degrees Celsius and 5%carbon dioxide, replacing the medium every two to three days.

Prepare the nucleofector solution and supplement mixture by combining 16.4 microliters of nucleofector solution with 3.6 microliters of supplement per tube. Then, re-suspend a cell pellet containing 100, 000 to 200, 000 cells isolated from the human cervical tissue sample in 20 microliters of the prepared nucleofector solution and supplement mix. Then add 300 nanomolar of either control or target small interfering RNA oligo to each tube containing the re-suspended cells and mix gently.

Carefully transfer 20 to 22 microliters of the nucleofection mixture into the electroporation cuvette vessels from the transfection kit. Gently tap the Nucleocuvette vessel to ensure the sample covers the bottom of the cuvette. Close the lid of the Nucleocuvette vessel and place the vessel into the retainer of a powered-on 4D-Nucleofector unit.

To initiate the nucleofection process, select the P4 primary cell process and set the pulse code to EA125 for each cuvette. After completion, carefully remove the Nucleocuvette vessel from the retainer and incubate the Nucleocuvette vessel at room temperature for 10 minutes to allow the cells to stabilize. Next, open the lid of the Nucleocuvette vessel and add 180 microliters of 37 degrees Celsius pre-warmed organoid culture medium to each cuvette.

Gently transfer the cell suspension to a new 1.5 milliliter tube for re-suspension. Finally, take a volume of the cell suspension containing 500 to 2, 000 cells, re-suspend in organoid culture medium to a final volume of 50 microliters and culture the organoids as demonstrated previously. Human cervical organoids grew up to 500 micrometers in diameter from single cells over two weeks.

Hematoxylin and eosin staining clearly revealed the formation of stratified squamous epithelium in the organoids. Immunofluorescent staining revealed that the basal layer was located in the outer rim of the organoids surrounding the super basal layers. Cervical organoids could be passaged at least 10 times over six months.

After electroporating the human cervical epithelial cells with siRNA oligos against AKT, about 97.3%of cells were viable. Simultaneous silencing of AKT1 and AKT2 effectively silenced both genes and interfered with cervical organoid formation. This protocol enables quantitative evaluation of drug and gene function in human cervical stem cells, facilitating mechanistic studies of cervical development and disease.

Peeling or scraping of small pieces of cervical epithelia in the process of single cell dissociation can be technically challenging for beginners. Histologic analysis such as H&E staining and immuno staining, transcriptomic or proteomic analysis and functional perturbation assays can be done following this procedure.