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The overall goal of the following experiment is to generate three dimensional typic models of early stage ovarian cancer development. First, generate H turt immortalized cell lines derived from primary, normal ovarian epithelial and fibroblast tissues. Then to recreate the in vivo architecture and complex cell cell interactions present in the ovary co-culture, epithelial and stromal cell lines in three dimensional S spheroid models proceed to fix and stain the organoid cultures and analyze expression of specific markers in order to study molecular features of stromal epithelial interactions.

Results obtained show how alterations in the stromal cell compartment of 3D typic models can influence the phenotype of co cultured epithelial cells. Three dimensional typic models can help answer key questions in the ovarian cancer research field, such as exploring the origins of this disease and elucidating the role of the stromal microenvironment in tumor initiation and development. Applications of these techniques extend towards disease diagnosis and therapy compared to other 3D culturing methods.

This technique can be applied to a wider range of cell culture volumes and downstream applications. For example, 3D homo and typic cultures can be used for in vitro drug testing. This approach can also be applied to other model systems, such as in the study of other solid tumor types or in benign disease.

Transport the fresh ovarian tissue to the cell culture laboratory in immortalized normal ovarian fibroblast growth medium supplemented with extra antibiotics and antics. Wash the sample three times with five milliliters of PBS to remove any epithelial cells that remain loosely attached to the tissue. Next, transfer the sample plus PBS into a clean P 100 culture dish Using sterile instruments, transfer the tissue into a second dry P 100 clean culture dish.

Cut the tissue into 0.5 square centimeter pieces and transfer each piece into a separate P 100 dish. Continue to cut the tissue into pieces less than one square millimeter in size. Then add 20 milliliters of INOF GM media and culture Monitor for cell growth using phase microscopy.

Confirm that the cells adhere to the dish within 24 hours. Monitor for cell growth using phase microscopy Cells usually adhere to the dish within 24 hours After a week. Remove any non-adherent tissue by aspiration, replenish the media and thereafter refeed cultures twice a week within one to three weeks when cell colonies are about 500 microns in size.

Isolate the clones using trips and coated cloning discs. In order to confirm 100%fibroblastic cells. Plate a sample of the cell line onto glass cover slips and perform immunofluorescent staining for a fibroblastic marker, an epithelial marker, and an endothelial cell marker.

One day prior to retroviral transduction of H Turt passage, the normal ovarian fibroblasts cell isolates from a single P 100 dish into three P 100 dishes. Pre-made viral sup natan can be purchased or produced in-house using standard protocols for cot transfection of HEC 2 9 3 T cells. To prepare the poly Hema solution, weigh out 1.5 grams poly hema, and place into a sterile bottle.

Add 95 milliliters molecular biology, grade, absolute ethanol, and five milliliters cell culture grade distilled water. Place the poly Hema solution in a water bath at 65 degrees Celsius until fully dissolved. Coat the tissue culture dishes with freshly made poly Hema solution.

Air dry the cell culture dishes inside the laminar flow hood. Gentle rocking on a rocking platform can be used to ensure even coating of larger size dishes or flasks. If the polyus solution dries too quickly, the surface will not be smooth.

If desired, pretreat the fibroblasts prior to 3D culture, for example, for induction of senescence, pretreat the cells with sub lethal doses of hydrogen peroxide. Recover and expand the INOF and epithelial cell cultures in vitro to prepare enough cells for establishing 3D heterotopic steroids. After a five minute PBS wash, add 15 milliliters of medium to the poly hema plate.

Meanwhile, add trypsin to the immortalized normal ovarian fibroblast cells to detach them from the culture dish. After three to five minutes, neutralize the trypsin with an equal volume of culture media and harvest the cells by centrifugation. Resuspend the cell pellet in five milliliters medium and mix well by pipetting up and down or vortexing gently.

Then use a 10 microliter sample to enumerate viable cells using trian blue. Now add 4 million INOF cells to the medium already dispensed into the poly hema coated plate. Make up the culture media volume to 20 milliliters and incubate at 37 degrees Celsius, 5%carbon dioxide.

Monitor the cellular aggregation into multicellular steroid by phase microscopy to refeed the S spheroid cultures every two to three days. Rest the plates at an angle on a pipette. Allow the spheroids to settle and carefully remove about 16 milliliters of the exhausted media with a five milliliter pipette.

Add 16 milliliters of fresh medium to the culture and return to the incubator On day seven. Create hetero typic cultures by adding epithelial cells to the fibroblasts phe. First, prepare suspensions of ovarian epithelial cells by trypsin as described earlier after enumerating cells.

Wash once in INOF GM medium to prevent any growth factors from being carried over from the epithelial growth media. After replenishing medium of the fibroblast OID cultures, add the epithelial cells at a ratio of four to one fibroblasts to epithelial cells culture. The heterotopic OIDs in INOF GM refeeding typic cultures every two to three days for an additional 14 days.

For immunohistochemistry, fix the PHE with neutral buffered formin, pellet the PHE and replace the formalin with 70%ethanol. Proceed to process into paraffin using standard protocols used for human tissues. Paraffin embedded steroids can be sectioned and stained with h and e of specific molecular markers using immunohistic chemistry.

Alternatively, wash the steroids in PBS and fix in 4%para formaldehyde. Then embed the samples in OCT medium and snap freeze for cryostat sectioning and immunofluorescent staining for analysis by flow cytometry. Wash the steroids in PBS and dissociate by trypsin or Accutane digestion at 37 degrees Celsius.

Complete dissociation of steroids into a single cell suspension can be promoted by pipetting the solution up and down with a one milliliter pipette tip. Take care not to oversize the cells, Neutralize the reaction with an equal volume of culture medium and remove cell clumps by passing the suspension through a 40 to 70 micron cell strainer. Then pellet the cells and wash with PBS and enumerate resuspend the desired number of cells in facts buffer and stain according to manufacturer's instructions In our laboratory, we have partially transformed ovarian epithelial cells and co cultured these cells with normal and senescent ovarian fibroblast to mimic early stage ovarian cancer development.

In a premenopausal ovary Immunofluorescent staining of h hert immortalized normal ovarian fibroblasts show that both primary normal ovarian fibroblasts and immortalized normal ovarian fibroblasts expressed fibroblast specific protein, but they do not express cytokeratin seven and express menton. Normal ovarian fibroblast cells cultured alone in 3D form PHE display a spindled morphology and abundant extracellular matrix. Here, normal ovarian fibroblasts were exposed to hydrogen peroxide to induce senescence over seven days and then cultured with partially transformed ovarian epithelial cells for 14 days.

To distinguish the two cell types, the resulting steroids were immuno stained for mim one as a proliferation marker and pan cytokeratin as an epithelial marker. Quantification of the data suggests that the aging microenvironment promotes neoplastic features of partially transformed epithelial cells, whereas a normal microenvironment inhibits neoplastic progression, histological features of normal and malignant cells that are not detectable when the cells are cultured as monolayers are restored. When the cells are transitioned into spheroid culture, for example, histological features of clear cell ovarian cancers can be detected in 3D cultures of clear cell ovarian cancer cell lines, but not in monolayer cultures of the same cells.

After watching this video, you should have a good understanding of how to set up three dimensional typic cultures with which to study the interactions between epithelial and stromal cells. During early tumor genesis. When setting up a 3D culture, it is important to adjust cell numbers according to the downstream application, allowing for some loss of material during processing.

Once mastered, 3D cultures can be set up in one hour following this procedure. Other methods such as flow cytometry combined with gene expression microray profiling can be performed to analyze the global gene expression changes that occur in 3D typic cultures but are not detected under monolayer culture conditions. This will enable dissection of the bidirectional signaling pathways that occur specifically under a 3D core culture.Microenvironment.