Selection of the membrane for centrifugal ultrafiltration and protein profile analysis of human primary keratinocyte secretome

The potential of keratinocyte secretome as a cell-free regenerative medicine for wound healing necessitates the optimization of its concentration. This study aimed to select an appropriate molecular weight cutoff (MWCO) membrane for centrifugal ultrafiltration of the conditioned medium from human primary keratinocyte cell culture. Three membranes with MWCOs of 3, 10, and 30 kDa were tested at centrifugation speeds of 4,000´g and 14,000´g. The 30 kDa membrane proved unsuitable as all the solution permeated, leaving no retentate. At the lower centrifugation speed, protein loss due to adsorption to the membrane was significant, resulting in suboptimal concentrations. In contrast, using the 10 kDa membrane at 14,000´g achieved a near two-fold increase in protein concentration. The concentrated secretome extracellular vesicles ranged between 20 - 200 nm. The protein profile analysis using mass spectrometry confirmed the presence of essential growth factors for wound healing, including vascular endothelial growth factors (VEGF), fibroblast growth factors (FGF), and epidermal growth factors (EGF). Our findings demonstrate that a 10 kDa membrane is optimal for obtaining a concentrated secretome rich in vital wound-healing components. This membrane will be used for further study of the large-scale production of the keratinocyte secretome using tangential flow filtration.