Primary Cell Isolation and Culture
In brief, AMDC was obtained using enzymatic digestion and by mechanical means20, harvested from excised uterus with AM completely which have not used hormone therapy in the past three months. Ectopic adenomyotic samples were collected from hysterectomy specimens by cutting the obvious adenomyotic tissue at least 2.5 mm below the endometrial-myometrial interface. The presence of AM was confirmed by frozen section and histopathologic examination. Each specimen is divided into two parts, one put in 10% formaldehyde (Biosharp, BL539A, Chongqing, China) for pathological examination, as shown inFigure 1 , the other was placed in put in with sterile D-Hanks (without calcium, magnesium, with phenol red) (Solarbio, H1045, Beijing, China) several times to wash away impurities and blood stains, put it into fresh tissue protection solution with 10% penicillin-streptomycin (Solarbio, P1400, Beijing, China) antibiotics and then transferred to the laboratory. Removal postoperative medical examination did not meet the pathological changes of AM timely. The tissues were gently cut into small pieces (1 mm3) and added 4 times the volume of 2mg/mL collagenase IV (diluted with D-Hanks) ((Solarbio, C8160, Beijing, China), digested in a 37°C water bath for 4 hours, shaking the tissue every 10 minutes during the digestion process to make the tissue and enzyme fully mix and contact. Adding 3-5 times the volume of 10% Fetal Bovine Serum (UY; Lonsera S711-001S, South America) after digesting to chyle for completing medium to neutralize the digestive activity of collagenase. The dispersed cells were filtered through 70-μm and 40-μm (Becton Dickinson, Franklin Lakes, NJ, NY, USA) filter screens respectively to remove the undigested tissue pieces. The AMDC was collected by centrifugation (Baiyang, Beijing, China) at 1000 rpm for 5 min and washed 2–3 times with Phosphate-Buffered Saline solution (PBS) sterile.
The isolated AMDC was cultured in medium solution completely, using 35.6mL DMEM/F-12 (1:1) basic (1×) (C11330500BT, Gibco, Shanghai, China) with 10% UY 4 mL and 1% penicillin-streptomycin antibiotics 400μl. These cells in culture dishes were incubated at 37 °C in a humidified atmosphere of 5% CO2-95% air (Germany,Memmert). Cells from the sixth to the tenth passages were used for further experiments.