An Efficient Method for Extracting Human Fallopian Tube Epithelia for Single-cell Analyses

Our research focus is understanding and combating chemo resistance in ovarian cancers. We aim to discover mechanisms and cell subpopulations driving resistance and novel targets to eradicate these aggressive populations.

Single cell RNA sequencing facilitates characterization of subpopulations in heterogeneous ovarian tumors. We leverage non-cancerous fallopian tube epithelial cells as a normal origin cell for ovarian cancer analyses.

The efficient isolation of fallopian tube epithelial cells into an enriched single-cell suspension will aid in further understanding high-grade serous ovarian cancer disease initiation. This mechanical and enzymatic digestion technique offers a more efficient process for extracting an enriched single-cell suspension of viable fallopian tube epithelial cells.

[Narrator] To begin, collect fresh human fallopian tubes in a 15 or 50-milliliter tube containing dissociation media and place the tube on ice. Transfer the fallopian tubes into a sterile Petri dish containing fresh dissociation media and place the sample under a dissecting microscope. Now, using fine point forceps and Vannas-Tubingen scissors, dissect away fat, connective tissue, and any vessels surrounding the tubes. Then, cut through the coronal plane of the fallopian tubes with scissors to form small cylinders with three to five millimeters diameter. Use fine=point forceps to stabilize the tissue while cutting. Carefully aspirate the dissociation media from the dish containing the tissue pieces. Wash the tissue pieces two times with two milliliters of cold 1x PBS and lightly rotate the plate. After aspirating the PBS, incubate the tissue pieces for five minutes in cold five millimolar EDTA at room temperature. Suspend the tissue fragments in cold 1% Trypsin Hanks Balanced Salt Solution and incubate at four degrees Celsius for 40 minutes. Aspirate the 1% Trypsin Hanks Balanced Salt Solution and wash the tissue pieces twice in cold dissociation media to inactivate the trypsin. Incubate the tissue fragments in two milliliters of dissociation media with 0.4 milligrams of DNase for at least 30 minutes at room temperature. Under a dissecting microscope while still in DNase media, hold a fragment of the fallopian tube with forceps so the lumen is parallel to the dish bottom. Use forceps to repeatedly press on the tissue to expel epithelial cells from the lumen. Confirm the release of a halo of cells around the tissue. Transfer the media-containing cells into a sterile 15-milliliter tube. Wash the Petri dish twice with dissociation media and combine washes in the same tube. Discard the remaining stromal tube fragments. To harvest the cells, centrifuge them at 500g for five minutes. Aspirate the supernatant and resuspend the pellet in one milliliter of dissociation media. Now, mix 10 microliters of cell suspension with an equal volume of trypan blue. Pipette 10 microliters of the mixture into a chamber counting slide and insert it into a cell counter to determine the cell count. Resuspend the cells in nine milliliters of dissociation media containing collagenase type one and DNase. Incubate at 37 degrees Celsius for 30 to 45 minutes in an orbital shaker set to 200 RPM. To harvest the cells, centrifuge at 500g for five minutes. After aspirating the supernatant, resuspend the pellet in five milliliters of dissociation media to wash off collagenase. Pass the cell suspension through a 100-micrometer cell strainer into a 50-milliliter tube and centrifuge the filtrate at 500g for five minutes. If the cell pellet appears red, prepare an RBC lysis buffer to lyse the red blood cells. Aspirate the media from the pellet, add five milliliters of the diluted RBC lysis buffer, and incubate the sample on ice for three minutes. To stop RBC lysis, add 45 milliliters of 1x PBS to the tube. Centrifuge the tube at 500g for five minutes. After harvesting and resuspending the cells in one milliliter of dissociation media, mix 10 microliters of the cell suspension with an equal volume of trypan blue. Pipette 10 microliters of the mixture into a chamber counting slide and insert it into the cell counter. Flow cytometry analysis confirmed the isolation of a viable and enriched epithelial cell population from fallopian tube tissue with an average viability of 82%. After getting out CD45 positive immune cells, EpCAM positive epithelial cells comprised an average of 80% of the sample, while CD10 positive stromal cell contamination was minimal at 7.8%. Isolated epithelial cells formed adherent cobblestone-like clusters in 2D culture after four to six days of plating, confirming epithelial identity. Immunochemistry revealed that most cultured cells expressed EpCAM and PAX8 with only a few vimentin-positive cells. Ciliated cells were present in culture.