Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

The overall goal of this experiment is to create an organotypic model that mimics the peritoneal microenvironment to better our understanding of ovarian cancer biology. This method can help answer key questions in the ovarian cancer field, such as how to interactions between cancer cells and the microenvironment contribute to disease pathogenesis, and how can potential inhibitors of these interactions be tested. The main advantage of this technique is that actual primary human cells that form the mesothelium line surfaces of the peral cavity are used in the ovarian cancer cell co-culture.

Though this method can provide insight into ovarian cancer, it can also be applied to the study of other cancers that disseminate throughout the peritoneal cavity, such as gastric, pancreatic, and colon cancers. Upon acquisition of a surgical human omentum specimen immediately immerse the sample in room temperature PBS. Within two hours of the immersion, transfer the omentum to a 50 milliliter conical tube containing 20 milliliters of fresh PBS transfer the remaining PBS wash containing the primary human mesothelial cells or HPMC and red blood cells into a new 50 milliliter conical tube and spin down the cells.

Then pour the tube contents into a 10 centimeter sterile culture dish and use two scalpels to mince the tissue into five cubic millimeter pieces. To isolate the primary human mesothelial cells or HPMC, transfer the omentum pieces into a 50 milliliter conical tube and wait one minute. For the solid pieces to float to the top, the HPMC will settle at the bottom of the tube with the red blood cells.

Use a pipette to transfer the PBS wash containing mesothelial cells and red blood cells and into the tube containing the pelleted cells and spin down the cells. Then add 20 milliliters of fresh PBS to the omentum and collect the PBS containing HPMC and RB C3 more times is just demonstrated to the original pelleted cells. After the final spin plate, the cells in a 75 square centimeter flask in 15 milliliters of full growth medium.

To isolate additional HPMC from the omentum sample, shake the remaining solid tissue in 20 milliliters of PBS at 200 RPM, and 37 degrees Celsius. After 10 minutes, let the sample rest for one minute to allow the solid tissue to rise to the top of the tube. Then collect the HPMC and RBC from the bottom of the tube as just demonstrated.

Now, spin down the cells from this secondary wash and plate them in a separate 75 square centimeter flask in 15 milliliters of full growth medium. To isolate any remaining HPMC, shake the tissue for another 10 minutes this time in a mix of PBS and 10 milliliters of tripsin EDTA and plate these HPMC and RBC in a 75 square centimeter flask then incubate the cultures at 37 degrees Celsius and 5%carbon dioxide in a humidified environment, feeding the plated cells with 15 milliliters of fresh, full growth medium on days three and five without removing the spent medium. The HPMC can be identified by their cuboidal shape and their expression of cytokeratin eight and menton.

To isolate the normal omentum fibroblasts, treat the remaining minced omentum tissue with 10 milliliters of freshly prepared 10 x collagenase type three solution diluted in serum free medium for six hours with shaking at the end of the incubation period, the digested tissue will have become opaque with the solution containing some fibrous debris, centrifuge the normal omentum fibroblast suspension, and then culture. The cells in 13 milliliters of full growth medium. After 24 hours, replace the old medium with 15 milliliters of fresh full growth medium.

The normal omentum fibroblasts can be identified by their flat elongated shape, their expression of vimentin and their lack of cytokeratin aid expression. To release the normal omentum fibroblasts from culture, rinse the culture flask with 10 milliliters of PBS, followed by three milliliters of trypsin for no more than five minutes. When the cells have detached, neutralize the trypsin with at least three times the volume of full growth medium, and transfer the cells to a 50 milliliter conical tube.

Spin down the cells and re suspend the pellet in five milliliters of full growth medium. Then count the cells and dilute them to a two to four times 10 to the third fibroblast per 100 microliters of full growth, medium concentration. Next, add 0.5 micrograms per 100 microliters of rat tail collagen, one to the cells and plate 100 microliters of cells per well into a black clear bottom 96 well plate.

Then transfer the plate to a cell culture incubator for at least four hours or until the cells adhere to the plate surface, while the normal momentum fibroblasts are settling. Detach the HPMC from their culture flasks and dilute these cells to one to two times 10 to the fourth HPMC per 50 microliters of full growth medium. Then add 50 microliters of HPMC per well to the attached normal omentum fibroblasts, and return the plate to the cell culture incubator for at least 18 hours before experimental.

Use the next day seed, the 3D ORGANOTYPIC co cultures with GFP expressing ovarian cancer cells from an 80 to 90%confluent culture at the appropriate dilution for the desired downstream assay, the RGD peptide and blocking antibodies against the alpha five, beta one and alpha five. Beta three. Integrins inhibit the adhesion of ovarian cancer cells to the organotypic culture while the RAD peptide and control and beta four integrin antibodies do not.

The RRG D peptide and blocking antibodies against alpha five and beta one integrin also inhibit ovarian cancer cell proliferation on the organotypic culture while the RAD peptide and control alpha five, beta three and beta four integrin antibodies do not. Further the RGD peptide and blocking antibodies against alpha five and beta one integrin inhibit ovarian cancer cell 3D ORGANOTYPIC culture invasion. While the alpha five Beta three integrin antibody enhances this process as expected from the adherence and proliferation assay data, the RAD peptide and control and beta four integrin antibodies also exhibited no effect on cancer cell invasion.

Once mastered, this technique can be completed in seven to 12 hours if it is performed properly. While attempting this procedure, it's important to remember to always use a biological safety cabinet and the appropriate protective equipment, including a lab coat and gloves following this procedure. Other methods like adhesion, invasion and proliferation assays can be performed to answer additional questions about cancer cell interactions with the microenvironment after its development.

This technique paved the way for researchers in the field of ovarian cancer to explore how the microenvironment influences cancer cell interactions with human peritoneal cavity cells. After watching this video, you should have a good understanding of how to isolate primary cells from the human momentum and how to build an organotypic culture of the peral lining.