The overall goal of this procedure is to establish primary endometrial stromal cell cultures from resected hysterectomies. This is accomplished by first distinguishing and separating uterine layers to isolate a section of endometrial tissue. The second step is to choose a cell isolation technique between the scraping and tryin methods when utilizing the scraping method.
Mechanical force and scraping are used to separate and dissociate endometrial stromal cells. Alternatively, when utilizing the trypsin method, the enzymatic activity of trypsin is used to separate and dissociate endometrial stromal cells. Ultimately, microscopy is used to observe cell growth and morphology, while other techniques discussed in the manuscript are used to verify endometrial stromal cultures.
This method can help answer key physiology questions in the reproductive field phenomena such as the reproductive cycle and menstruation Hysterectomy specimens used in this manuscript were collected in concordance with a university IRB approved ethics protocol numbered I-R-B-H-S-R number 1 4 4 2 4. Maintain patient-derived tissue in a 50 milliliter tube containing RPMI or DMEM with high glucose at four degrees Celsius for no more than 24 hours before the sample is processed. When ready to begin, wash the tissue sample three times with one XPBS and discard the solution between each wash.
Then add four to 10 milliliters of growth media supplemented with 10%FBS and 1%penicillin streptomycin to the tissue sample. Also, add a fungi zone to a final concentration of 0.25 micrograms per milliliter and incubate for 30 minutes. Next, discard the growth media and wash the tissue twice with one XPBS.
Place the tissue on a six centimeter cell culture plate and identify the endometrium layer using these images as a guide. Then separate the endometrium and save the other layers of tissue for future use. Use a scalpel or razor blade to transect the tissue into small pieces while scratching onto the cell culture dish.
The scratches facilitate the attachment of the emerging primary endometrial stromal cells. Next, gently add two milliliters of growth media to the scratched dish. At this stage, tissue fragments will be visible and immobilized from the scratching motion.
Place the dish in a designated cell culture incubator separate from other cell lines to avoid contamination. Monitor the cells daily using a light microscope. Look for small populations of cells who emerge from the sliced tissue by day two or three every three days.
Gently remove the old media wash cultures with one XPBS and then add two milliliters of fresh growth media. Begin adding four milliliters of growth media. Once the proliferation rate increases during the wash steps aspirate the remaining pieces of tissue.
New colonies are unlikely to emerge from the tissue. After one week of growth passage, the cells using 0.05%trypsin. When the cells reach between 75 and 80%co fluency then freeze down one plate of cells.
Once two or three plates are maintained, primary cells cannot be passaged indefinitely. To begin tripsin mediated isolation, place a small piece of endometrial tissue in a conical tube with two milliliters of 0.25%Trypsin supplemented with 0.03 milligrams per milliliter. Can mycin then incubate the tissue on a rotating platform at 37 degrees Celsius for 30 minutes.
Next, briefly vortex the tissue and then centrifuge at 200 to 400 times G for two minutes, discard the supernatant and add fresh trypsin. And can mycin then incubate the tissue at 37 degrees Celsius for one hour while rotating. After the incubation, vortex the tissue for five to 10 seconds.
Then add two milliliters of growth media containing 10%FBS and 1%penicillin streptomycin. To deactivate the trypsin, centrifuge the cells at 200 to 400 times G for two to three minutes. Then discard the supernatant and resuspend the cell pellet.
In two milliliters of growth media plate the cell suspension onto a six centimeter cell culture dish and scrape the plate to enhance attachment of the primary cells. Monitor cell growth daily with a light microscope and change the growth media every two to three days. Once cells have reached a co fluency of 75 to 80%use 0.05%or 0.25%trypsin to passage them isolating endometrial stromal cells.
Using the scraping method generates clusters of early emerging cells. Along the scalpel induced scratches, the endometrial stromal cells can be identified by their fibroblast morphology. The trypsin method has a longer preparation time than the scraping method, but viable cells are observed earlier.
The tryin method also generates cells with both clustered and dispersed growth patterns. A tissue specific marker for endometrial stromal cells has not yet been discovered. One way endometrial stromal cells are identified is by measuring the loss of a marker.
For epithelial cells such as cytokeratin. Lack of cytokeratin demonstrates an absence of endometrial epithelial cells. The loss of cytokeratin staining observed in passage five compared to passage two, confirms the absence of epithelial cells.
Functional evidence for the presence of endometrial stromal cells can be provided using a decellularization assay. The increase in prolactin and insulin-like growth factor binding protein one in response to treatment with CAMP and hydroxy progesterone acetate indicates successful establishment of an endometrial stromal culture. After watching this video, you should have a good understanding of how to establish primary endometrial stromal cell cultures from resected hysterectomies.
