Deoxynuclease 1

Isolation, culture, and purification of ESCs were explained earlier.³² Briefly, endometrial tissues obtained from patients and controls were cut into 1 mm³ pieces and digested in DMEM‐F12 (Gibco) containing penicillin (100 U/ml) and streptomycin (100 μg/ml; Gibco), collagenase type I (2 mg/ml; Sigma‐Aldrich) and deoxynuclease I (300 μg/ml; Takara) in a humidified 5% CO₂ at 37°C for 2 h with intermittent vortexing every 15 min. Following the removal of the undigested tissue using 100 μm mesh (BD Biosciences), cells were cultured in DMEM‐F12 (Gibco) medium supplemented with 10% CS‐FBS (Sigma‐Aldrich) and 1% pen‐strep antibiotic (Gibco) in a humidified 5% CO₂ at 37°C for 24 h. After the removal of non‐adherent cells by washing with warm medium, adherent stromal cells were allowed to multiply. To evaluate the purity of ESCs, immunofluorescent staining and flow cytometry analysis were used. These cells were characterized as a panel of vimentin⁺, nestin⁺, cytokeratin⁻, CD10⁺, CD44⁺, CD73⁺, CD105⁺, CD34⁻, and CD45⁻ cells.³³

Fresh endometriotic tissues were collected in sterile Dulbecco’s modified Eagle’s medium (DMEM) –F12 (Gibco, Grand Island, NY, USA) containing antibiotics and immediately transferred in cold chain to the laboratory, where minced into small pieces and digested at 37 °C for 120 min in DMEM/F-12 containing 100 U/ml penicillin and 100 μg/ml streptomycin, 2 mg/ml of type I collagenase (Sigma-Aldrich, St Louis, MO, USA) and 300 μg/ml of deoxynuclease I (Takara, Tokyo, Japan) with intermittent vortexing every 15 min (20 (link)). The obtained ESCs were then cultured and allowed to propagate not beyond passage three. Some samples were lost due to contamination or absence of enough cell growth especially in case of EESCs. Finally, cells from 13 eutopic and 11 ectopic endometrial tissues of endometriotic patients and 11 eutopic endometrial tissue from non-endometriotic patients were used in this study. Purity of isolated ESCs was assessed by flowcytometric analysis using a panel of antibodies. The ESCs were characterized as CD10⁺, CD73⁺, CD105⁺, CD44⁺, CD34⁻ and CD45⁻ cells.

Lungs were isolated from fat-1 transgenic and wild type mice and washed with saline to remove blood cells. Isolation and culture of pulmonary fibroblasts were performed using methods described previously [33] (link). Lung tissues were minced into small pieces and incubated in DMEM (Gibco, NY, USA) containing type I collagenase (0.25%; Sigma-Aldrich, St Louis, MO, USA) and deoxynuclease I (15 U/ml; TaKaRa, Tokyo, Japan) for 120 min at 37°C. The resultant dispersed cells were separated by filtration through nylon cell strainers (70 µm, BD, Franklin Lakes, NJ, USA). Fibroblasts in the filtrate were collected, placed into 10 cm dishes in DMEM containing 10% FBS, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 mg/l amphotericin B, and incubated for 7–10 days. Fibroblasts were purified from other cell population by differential adhesion and serial passage.
Confluent fibroblasts and TC-1 cells were trypsinized and resuspended in DMEM containing 10% FBS, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 g/ml amphotericin B and 1×10⁵ cells/ml cells of each cell type were plated together in 12 well culture plates. Co-cultures were incubated at 37°C 5% CO2 in a humidified atmosphere for 24 hours. Homotypic cultures served as controls.