Department of Biomedical EngineeringA three-dimensional spheroid culture system to harvest conditioned medium (CM) from human bone marrow-derived mesenchymal stem cells (hBM-MSCs) with high efficiency were introduced and investigated the therapeutic effects of hBM-MSC-CM on erectile dysfunction (ED). In order to harvest CM with higher protein concentration than CM harvested by conventional method in 2D culture system, a variable parameter study considering the culture conditions such as spheroid diameter and centrifugation time was performed. As a result, it was confirmed that CM harvested from 3D spheroid culture system had much more proteins than those cultured in 2D environment. In addition, the total protein concentration in CM increased in proportion to the centrifugation time using a centrifugal filter. Additionally, ELISA and secretome assay showed that CM contained growth factors that help in angiogenesis such as angiogenin (ANG) and vascular endothelial growth factor (VEGF), and in nerve regeneration like glial cell-derived neurotrophic factor (GDNF), nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF). Also, it was identified that angiogenic effects of CM contributing to tube, node and mesh formation of endothelial cells was proportional to the concentration of CM in vitro tube formation assay. Then, histomorphometric studies such as immunohistochemistry and immunofluorescence were performed using the cavernous nerve (CN) injury rat model. In result, histomorphometric analysis showed that neural regeneration and vascular regeneration increased along with CM concentration. To sum up, we established an efficient method to obtain hBM-MSC-CM containing high concentration of growth factors, which is expected to be one of the promising methods of regenerative medicine for treating ED disease.clos
