Collagenase 1

Isolation and culture of ADSCs was partially performed using the standard protocols of Bura et al (21 (link)) and Guo et al (22 (link)). Permission and a signed consent to participate from the Institutional Review Board of Medical Science (Jinan University, Guangzhou, China) was obtained prior to the collection of 100 ml abdominal subcutaneous fat from a 65-year-old patient in November 2016. The adipose tissue was digested with 0.2% collagenase I (Biochrom GmbH, Berlin, Germany) for 30 min at 37°C, centrifuged for 10 min at 300 × g (RT) and filtered with a 100 µm mesh filter (neoLab Migge GmbH, Heidelberg, Germany). NaCl (0.3%) was used to remove the red blood cells prior to transfer of cells into culture medium including Dulbecco's Modified Eagle's Medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal calf serum (Gibco; Thermo Fisher Scientific, Inc.), 1% 100 U/ml penicillin and 100 µg/ml streptomycin, and culture in 5% CO₂ at 37°C. The culture medium was changed at 3 day intervals. Primary cells were cultured for ~10 days and defined as ‘passage 0’. ADSCs of passage 2 which were cultured at culture medium and passaged twice, then prepared for subsequent experimentation.

Uterine horns were cut longitudinally and endometrial tissues were subjected to enzymatic digestion, according to a procedure described previously by Bodek et al. [8 (link)]. Briefly, to separate stromal tissue from epithelial cells, endometrial tissues were digested with 1 IU/mL dispase (Sigma-Aldrich) for 75 min at 37°C in Hanks' balanced salt solution (HBSS) at pH 7.4 (Sigma-Aldrich), and then filtered through nylon mesh. The epithelial cells were discarded, and the remaining endometrial tissue was digested for 80 min in 0.06% collagenase I (Biochrom) in HBSS supplemented with 1% bovine serum albumin (BSA; Sigma-Aldrich). The endometrial stromal cell suspension was filtered and collected by a series of three centrifugations (218g, 10 min).
The pelleted cells were suspended in Dulbecco's modified Eagle's medium (DMEM/F-12; Life Technologies) containing 10% fetal bovine serum (FBS), 100 IU/mL penicillin, 100 μg/mL streptomycin, and amphotericin B, and seeded on 100-mm, tissue culture-treated Petri dishes at 37°C in a 5% CO₂ and 95% air atmosphere (P = 0). The same volume of endometrial tissue was always processed, although cells were not counted before initial seeding. After 18 h the nonadherent and dead cells were removed and fresh medium was added. The primary cultured endometrial stromal cells (P = 0) were used for further experiments.