This procedure delineates propagation of normal ovarian and od ductile epithelial cells as a three dimensional organ culture. First, dissect the ovary UCT fat pad and part of the uterus from a mouse. Then remove the bursa and dissect apart the vid ductile membranes with a pair of forceps and cut each ovary or UCT into two to four pieces termed organoids.
Finally encapsulate the organoid in an alginate droplet on a piece of mesh fiber. As OSC and TEC cultured in defined media retain normal cell characteristics in their native three-dimensional architecture. Immunohistochemistry on tissue sections, RNA analysis and protein analysis can provide insights into the impact of OSC and TEC in ovarian cancer.
The main advantage of this technique as compared to traditional two dimensional immortalized cellular culture is that the cells can be grown in contact with the underlying extracellular matrix and stromal cells without genetic immortal that could alter cellular pathways. This method can help answer key questions in the ovarian cancer field, such as defining early events in cancer formation and determining from which cell type serous ovarian cancers Arise. Though this method can provide insight into ovarian cancer, it can also be applied to other systems such as ovarian follicle culture and pancreatic beta eyelet culture.
Generally, individuals new to this method will struggle because removal of the bursa and uc membranes without damaging the ovaries and UCT is challenging. First, prepare a 0.5%solution of alginate in sterile PBS and heat to 37 degrees Celsius mixed by inversion or rocking. Then draw the alginate into a one milliliter syringe with a 25 gauge needle and to maintain a 37 degree Celsius.
Next, prepare a 10 millimolar calcium chloride solution for cross-linking the alginate upon tissue encapsulation and warm to 37 degree Celsius. Also, prepare one milliliter of culture medium per well in a 24 well plate for each experiment include various drugs, peptides, or viruses in the culture medium. When dissecting the organs from the animal, remove any fat or residual tissue.
Gently pull off the burs membrane surrounding the ovary and cut the ovary or UCT into four pieces by bisecting the organ to gently uncoil the oviduct tissue. Pull apart the connecting membranes and extend the ends of the tube in opposite directions. Using forceps.
Before cutting, place a single alginate droplet about two microliters onto a sterilized mesh fiber. Using sterile forceps, move a piece of organ into the alginate droplet and center the droplet to invert the droplet containing the organoid. Flip the mesh fiber and allow gravity to force the alginate into a hanging drop on the edge of a 10 centimeter Petri dish containing 10 millimolar calcium chloride solution.
Tap the forceps so that the drop falls into solution. Incubate for two minutes. Remove with a sterile spoon containing pores to release extra 10 millimolar calcium chloride and transfer into the culture medium culture for a defined time period.
For the solubilization of the alginate gel incubate the cultures in 3.4 milligrams per milliliter. Alginate lyase dissolved in ovitz is L 15. Media at 37 degrees Celsius for 30 minutes or until the gel is completely dissolved.
Then remove the tissue from the alginate lyase. Now to collect the OSE, incubate the organoid with 500 units of collagenase in ovitz ISS. L 15 media for one hour vortex in two second pulses on medium power for one minute, followed by one second.
Pulses for one minute. Let the organ pieces fall to the bottom of the micro centrifuge tube and pipette the supernat into a fresh sterile micro centrifuge tube centrifuge for three minutes at 3000 RPM to pellet the ovarian surface epithelium. Finally, to collect the tubal epithelium, cut the tube lengthwise and place on tissue culture plastic over three to five days.
Cells will crawl out of the tubals stroma and grow on the tissue culture plastic. Collect the alginate encapsulated bead from the culture medium at the selected time point recross. Link the alginate to stiffen the gel by dropping the culture into sterile 10 millimolar calcium chloride solution for two minutes.
Now, fix the alginate encapsulated organ ensuring that the gel stays solid and fully encapsulates the tissue during fixation. Cell types such as the OSC that do not grow well on plastic surfaces can be cultured. Using this method and facilitate investigation into the normal cellular processes or the earliest events in cancer formation.
Under the microscope, the bursa and the surrounding fat pad were removed and processed to alginate encapsulated organoids incubated. In culture medium. The OSC repaired the surface that was wounded from the incision with the scalpel as a function of time and distance.
Immunohistochemical analysis using a cytokeratin eight antibody indicates that ovaries cut in half encapsulate the ovary more completely as compared to ovaries cut into quarters or eighths. Tissue architecture, cellular morphology and specific cellular growth rates are easily monitored using paraffin sections. For example, a single layer of ovarian surface epithelium is observed after culturing in serum free MEM for seven days.
While addition of five micrograms per milliliter insulin for seven days induces hyper proliferation of the ovarian surface epithelium and formation of multiple layers of cells. Here, immunofluorescence of green fluorescent protein is visualized through the alginate gel with standard microscopy following CDNA transfection. While alginate organ culture provides for growth and analysis of ovarian surface epithelium and primary follicles, it should be noted that the mature secondary follicles fail to survive in this culture system.
Alterations in protein or RNA can be analyzed from the entire organ or from specific cell types using enzymatic degradation. Treatment of cultured ovarian organoids with collagenase leaves the underlying stroma intact while allowing for enrichment of ovarian surface epithelium in the cell pellet obtained following treatment. This technique can be done in one to two hours if performed properly.
While attempting this procedure, it's important to remember to maintain sterility at all times. Following this procedure, additional methods can be performed such as immunohistochemistry, western blotting, and transcription arrays to answer additional questions like how cell morphology and tissue architecture contribute to early transformative events. After watching this video, you should have a good understanding of how to perform ovarian and oviduct three-dimensional alginate culture.
