The overall goals of this procedure are to isolate and culture primary mouse endometrial, stromal and epithelial cells for their co-culture for the study of in vitro decidualization. This method can help answer key questions in the field of reproductive medicine such as what is the paracrine signaling between epithelial and stromal cells during important processes like decidualization and trophoblast invasion, and how is this signaling pathway regulated. These techniques can be used to study the process of decidualization in an epithelial and stromal co-culture system.
By using this system, you can evaluate whether signaling molecules secreted by one of the two cells can effect this process. Before harvesting the uterus, perform a vaginal smear examination to check the cycle phases of the animals. To select only mice in the estrus phase.
As characterized by the presence of corneal epithelial cells in the vaginal lavage. Next, use forceps and scissors to open the abdomen of the first estrus animal and reflect the intestines to the side to visualize both uterine horns. Grabbing one horn at the most distal end, under the fallopian tube, dissect the uterine tissue from the fallopian tube using dissecting forceps, and Dumont number five forceps to remove the adipose and connective tissues.
Then, remove the horn at the distal end, above the uterine body. After removing the uterine horns, place them in a 35 millimeter Petri dish with three milliliters of HBSS. When all of the uterine horns have been collected, transfer the dish under a stereo microscope, and use forceps to remove any residual adipose and connective tissues.
Then, using Dumont number five forceps, dissecting forceps and small spring scissors, open the uterine horns longitudinally to expose their lumens, and rinse the tissues in a new Petri dish containing fresh HBSS. Now, transfer all of the horns into a 15 milliliter tube containing pancreatin and trypsin. And incubate the tissues horizontally for 60 minutes at four degrees celsius and 50 rpm on an orbital shaker.
At the end of the hour, transfer the tube to the bench for 45 minute horizontal incubation without shaking, followed by a 15 minute horizontal incubation without shaking in a 37 degree celsius water bath. At the end of the water bath incubation, carefully decanter the supernatant and transfer the uteri into a Petri dish containing cold mouse endometrial epithelial cell medium for five minutes to inactivate the trypsin activity. Next, transfer the tissues to a 15 milliliter tube containing three milliliters of cold HBSS, and vortex the tissues for 10 seconds to release the epithelial sheets.
Then, rinse the uteri in a new Petri dish containing three milliliter of fresh HBSS, and collect two more epithelial sheet suspensions in two new 15 milliliter tubes as just demonstrated. After the third tissue vortex, transfer the uteri into a 15 milliliter conical tube containing mouse endometrial stromal cell digestion mix for a 30 minute incubation at 37 degrees celsius and 200 rpm. Recover the epithelial sheets by pipetting the three cell suspensions gently through a 100 micron nylon mesh to remove the tissue debris, followed by centrifugation of the resulting cells'suspension.
Re-suspend the pellet in 12 milliliters of mouse endometrial epithelial cell medium with thorough mixing, then allow the solution to settle for five minutes to separate the remaining mouse endometrial stromal cell by gravity. At the end of the sedimentation, use a five milliliter pipette to carefully remove the upper two milliliters of the supernatant, and centrifuge the collected cells. Then, re-suspend the mouse epithelial pellet in epithelial medium with gentle pipetting.
And plate the cells onto collagen coated cover slips at the appropriate density for the planned downstream analysis. After the epithelial cell isolation, continue with the stromal cells and remove the mouse stromal cell digestion tube with the uteri from the incubator. After 30 minute incubation, as just demonstrated, gently shake the tube by hand for 10 seconds, and transfer the uteri to a Petri dish containing three milliliters of four degree celsisus HBSS.
Rinse the tissues well, then add three milliliters of stromal cell medium to the digestion tube to inactivate trypsin activity, and transfer the uteri to a 15 milliliter conical tube containing three milliliters of fresh mouse endometrial stromal cell medium. Shake the tube of tissue gentle for 10 seconds. Then, transfer the uteri to a dish of cold HBSS, followed by their transfer to a new 15 milliliter tube of stromal cell medium.
After a third tissue rinse, and transfer, incubate the uterine tissues in fresh mouse endometrial stromal cell digestion mix for a 30 minute, 37 degree celsius incubation. At the end of the digestion, pass the cell suspensions from tube one to three and the trypsin tube through a 40 micron nylon mesh and rinse the mesh with an additional five milliliters of mouse endometrial stromal cell medium. Then, collect the cells by centrifugation.
And re-suspend the pellet in fresh mouse endometrial stromal cell medium for plating on polylysine coated cover slips at the appropriate density for the planned downstream analysis. The purity of the primary cell cultures can be assessed by the differences in vimentin and cytokeratin expression, as vimentin levels are high in mouse endometrial stromal cells, and low in mouse endometrial epithelial cells. While cytokeratin levels are high in mouse endometrial epithelial cells, and low in mouse endometrial stromal cells.
The mentin/cytokeratin double staining further illustrates the expression of the mentin by stromal cells, as well as the exclusive expression of cytokaratin by the epithelial cells. The induction of decidualization in the stromal cell cultures by the application of cyclic adenosine monophosphate, and MPA, reveals that prolactin MRNA levels are dramatically increased after five days of treatment compared to control stromal cell cultures. As the epithelial cells are difficult to visualize in side cell culture inserts, a viability assay could be performed immediately after a five day decidualization simulation, with a color shift from blue to bright pink indicating the presence of viable epithelial cells.
Following this technique, other methods such as blastocyst adhesion assays, can be performed in order to evaluate whether factors secreted by the epithelial cells in the presence of a blastocyst can induce stromal cell decidualization.
