Ovarian cancer remains a highly lethal disease with varying clinical presentations, multiple histological subtypes, and a wide range of genetic aberrations. Serous ovarian carcinoma is the most common and aggressive subtype, and recent studies suggest that a significant number of these tumors arise from the fallopian tube rather than the ovary. The aim of this procedure is to create ex vivo cultures of human fallopian tube cells where both secretory and ciliated cells can be co cultured in a manner that preserves their normal morphology, polarity and physiology.
This is accomplished by first collecting fresh human fallopian tube tissue samples and dissecting them into smaller pieces. Then the epithelial cells are dissociated from the fallopian tube tissue stromer. Next fallopian tube epithelial cells are isolated and other cell types are removed from the culture.
The final step is the seeding of fallopian tube epithelial cells onto collagen coated filters and growth in co-culture. Ultimately, results can be obtained that show the presence of both fallopian tube secretory and ciliated cells in culture through immunofluorescence microscopy or immunohistochemistry. The main advantages of this technique over existing methods like the use of ovarian cancer cell lines, is that both types of fallopian tube epithelial cells can be studied together in an exvivo co culture that preserves the morphology architecture and polarity of normal human fallopian tube epithelium.
Before human fallopian tube epithelial cells can be cultured. It is necessary to prepare collagen coated transwell filters to act as a substrate for epithelial cell culture. First, transfer 30 milligrams of human placental collagen into a BCO containing 50 milliliters of distilled water and a magnetic stir bar.
Then pipette 100 microliters of glacial acetic acid directly onto the collagen. Place the beaker onto a stirring hot plate and heat the solution to 37 degrees Celsius while stirring heat and stir the solution for 20 to 30 minutes. Ensure that the collagen is fully dissolved and that no collagen strands remain.
After the collagen has dissolved, add 450 milliliters of distilled water and then while working in a tissue culture hood filter sterilized through a 0.2 micron membrane. Transfer the solution to a 50 milliliter tube. Prepare collagen coated transwell filter membranes by first using forceps to place the filters into the wells of a 24 well plate, then pipette 50 to 100 microliters of collagen solution onto the top of the transwell filter and incubate overnight at room temperature.
The remaining collagen solution may be stored at four degrees Celsius after collection fresh fallopian tube fimbria are deposited in sterile PBS in a 50 milliliter centrifuge tube and immediately placed on ice in the tissue culture hood pipette 20 milliliters of chilled PBS into the tube. To wash the tissue carefully aspirate the PBS without disturbing the tissue and repeat the washer further two times. If the tissue is contaminated with blood, use sterile forceps to transfer the tissue to a sterile 10 centimeter tissue culture dish.
Then hold one fallopian tube fibril with sterile forceps and use a scalpel with a sterile blade to cut the fibril lengthwise to maximize the exposure of the epithelial cells. Open up the fibril with the forceps until it is almost a flat sheet. Then cut the sheet into small pieces of around three millimeters in diameter.
Use forceps to transfer the pieces into a 50 milliliter centrifuge tube containing 45 milliliters of chilled dissociation media. Place the tube on a shaking platform and rock gently at four degrees Celsius for 24 to 72 hours. See the written protocol for the procedure to check for optimal dissociation.
Check the amount of clumping and tissue viability represented by beating ciliated cells. Both single cells and some small clumps should be visible after optimal dissociation is achieved. Inactivate the pronase by adding five milliliters of FBS.
Then invert the tube two or three times to break up the cell clumps into smaller aggregates. Allow the tube to sit at room temperature for one to two minutes so that larger clumps of stromal tissue sink to the bottom of the tube. Then without disturbing the larger pieces of tissue, use a serological pipette to aspirate the media containing the epithelial cells and transfer to a clean 50 milliliter tube pipette.
50 milliliters of cold MEM without FBS into the 50 milliliter tube containing the tissue remnants. Invert the tube to collect any remaining epithelial cells and then allow the large clumps to settle. Once again, collect the media without disturbing the larger clumps of tissue and transfer to a fresh 50 milliliter tube centrifuge, both 50 milliliter tubes of epithelial cell, suspension of 1000 rotations per minute For five minutes following centrifugation, aspirate the media and discard.
Next Reese, suspend both cell pellets in five milliliters of Prewarm 37 degrees Celsius, USG media by gently pipetting up and down. Then transfer the cell suspension into a paria culture dish and incubate in the tissue culture incubator at 37 degrees Celsius and 5%carbon dioxide for one to three hours to remove fibroblasts and red blood cells from the epithelial cell suspension. During the incubation wash, the collagen coated filters with 100 microliters of PBS to remove excess collagen.
It is essential to avoid touching the membrane as any damage may affect the quality of fallopian tube ex vivo cultures. After the incubation time has elapsed, transfer the cell suspension to a 15 milliliter centrifuge tube. Then rinse the plate with five milliliters of USG media and add this solution to the 15 milliliter centrifuge tube.
Then centrifuge the cell suspension of 1000 rotations per minute for five minutes following centrifugation. Aspirate the medium without disturbing the cell pellet reus. Suspend the pellet in one to three milliliters of USG media depending on the size of the pellet.
Using a hemo cytometer count the number of epithelial cells and ciliated cells in 10 microliters of cell suspension. Epithelial cells appear non colum and have a distinctly dark cell membrane and live ciliated cells will be seen to be moving. Exclude red blood cells from the cell count following the cell count.
Add enough media to dilute the epithelial cells to 1.65 times 10 to the five cells per 100 liters. To plate the cells first pipette 500 microliters of USG media into the wells of a 24 well plate. Next place a collagen coated transwell filter into each well and pipette 100 microliters of the cell suspension on top of the collagen ENC coated trends will insert incubate the plate containing the cells in the tissue culture incubator for 48 hours.
After this time, carefully rinse the apical side of the insert with USG media and pipette 50 to 100 microliters of media on top of the membrane. Next, aspirate the media from the basal side of the filter. Insert and replace with fresh USG media.
Then return the plate to the tissue culture incubator. Change the basal media every two days during the period of culture. When the cultures have reached the desired co fluency for the particular application.
Aspirate the basal media from the wells and carefully wash the cultures with 200 microliters of PBS. Repeat the wash twice. Leaving the final volume of PBS in the wells.
The membranes are processed one by one. First aspirate the PBS from a single. Well then use forceps to remove the transwell membrane insert and flip the insert upside down.
Next, use a sterile scalpel to make straight cuts around the edge of the membrane. Then use forceps to remove the membrane from the insert in one piece. The filter can then be processed according to standard procedures for the desired application.
This bright field image taken at 10 times magnification shows the surface of the transwell membrane after coating with collagen here, another image taken at 10 times magnification shows fallopian tube epithelial cells cultured collagen coated inserts where they form an epithelial layer. The arrow indicates the presence of cellular debris which may adhere to ciliated cells. This field image taken at 20 times magnification demonstrates a healthy layer of fallopian tube epithelial cells in culture.
This fluorescent microscope image shows immunofluorescent staining of the fallopian tube epithelial cultures with anti PAX eight, A marker of secretory cells in red and dpi. A nuclear marker shown in blue Here, both PAX eight and DPI are seen to overlap in this merged image. This image illustrates immunofluorescent staining of the fallopian tube epithelial cultures with anti cell.
Two, a marker of ciliated cells in red and dpi, a nuclear marker shown in blue. Both cell two and DPI are seen to overlap in this merged image After this development. This technique has paved the way for researchers in the field of ovarian cancer to explore the role of the fallopian tube as a novel site for the origin of sero ovarian cancer.
