Isolation, Characterization, and Differentiation of Endometrial Stem Cells
Isolation and culturing of menstrual blood-derived stem cells was procured by various methods as mentioned below. Although there are variations in collection, isolation, and processing of MenScs according to various researchers, the overall phenomenon remains the same. Menstrual effluent was procured by the use of a sterile menstrual cup, the Divacup, that may be a silicone medical grade menstrual cup. Since we are aware most menstrual cell specimens will have a bioburden level, they are treated with antibiotics and kept cold. The medium used to isolate and culture and the antibiotics used by various researchers vary according to their protocol and the experimental set up. To collect the effluent, the menstrual cup is inserted into the vagina similarly to the process of inserting a tampon. The collection is mostly scheduled during the heaviest flow of the cycle, and the cup will remain in place for no more than 2–4 h, again according to the experimental set up. The volume collected may vary from 5 ml–15 ml. Once the menstrual effluents are procured, the menstrual cup is removed and the blood is transferred to the sterile media/PBS with antibiotics. Optimal transport after collection would be 24 h. The supernatant were evaluated for bacteria. The cells were washed twice and seeded in a culture flask. These cells displayed stromal cell morphology in the serum-containing growth medium in a flask and doubled in number every 24 h. The cells were grown in Chang’s (Irvine Scientific, Santa Ana, CA) Complete Media. Chang’s media for adhesion and proliferation were evidenced to be effective. Cells were assessed for time to adherence to flask, growth rate, and number of passages. Cells were subcultured by using TrypLE Express (Invitrogen, Carlsbad, CA), washed, and replated in complete media. After the cells were processed, they were tested for characterization.