Conditioned medium (CM) was collected from endometrial cells in the menstrual phase. Freshly isolated epithelial cells or stromal cells (30,000 cells) were cultured in growth medium in 6-well plate for one day then washed with PBS and replaced with 3 ml of growth medium. After 2-days in culture, the CM was collected, ltered sterilised and stored at -80 o C until experimentation.
To concentrate the secretory factors from endometrial niche cells, the epithelial or stromal cells were cultured in 1 ml of serum-free DMEM/F-12 medium. The secretory factors in the CM were concentrated (CCM) after 48 hour by centrifugation at 4000g for 20 minutes at 4°C using Amicon ultra-15 centrifugal lter devices (EMD Millipore) with a molecular weight cut off of 10 kDa. The amount of the concentrated protein derived from one culture well was considered as one unit. Epithelial or stromal CCM (1/3-unit) was added into the growth medium for eMSC culture. The CCM collected from cell free DMEMF-12 medium was used as control.
To concentrate the secretory factors from endometrial niche cells, the epithelial or stromal cells were cultured in 1 ml of serum-free DMEM/F-12 medium. The secretory factors in the CM were concentrated (CCM) after 48 hour by centrifugation at 4000g for 20 minutes at 4°C using Amicon ultra-15 centrifugal lter devices (EMD Millipore) with a molecular weight cut off of 10 kDa. The amount of the concentrated protein derived from one culture well was considered as one unit. Epithelial or stromal CCM (1/3-unit) was added into the growth medium for eMSC culture. The CCM collected from cell free DMEMF-12 medium was used as control.