Background & Aim Counterflow centrifugal (CFC) systems have been applied to cell manufacturing for the processes of cell selection and buffer exchange. The recently developed Rotea™ (Scinogy) is a CFC device that enables closed automated buffer exchange for small-scale cell manufacturing. However, the fluid dynamics within the processing chamber is complex and are influenced by a number of factors, including flowrate, centrifugal force, and buffer density. The impact of individual or compounded effects on cell quality is unclear. This study aims to determine the critical factors for mesenchymal stem cell (MSC) processing using the Rotea™. Methods, Results & Conclusion Methods We utilised a two-level full factorial Design of Experiment (DoE) approach to evaluate four parameters in the processing of umbilical cord-derived MSCs (UC-MSCs). These parameters included flowrate, centrifugal force-to-flowrate ratio (F/F ratio), serum concentration, and trypsin concentration. In each experiment, MSCs were subjected to one-hour of processing using a combination of high or low of each parameter to a total of 16 combinations, each combination was tested once. Live cell recovery, cell metabolism and function were determined using Trypan blue exclusion, MTS and IDO assays, respectively. Results The result of each test is expressed as a percentage to the unprocessed control. Live cell recovery was not correlated with any of the factors. High flowrate (Effect=-10.70, p=0.014) and high F/F ratio (Effect=-13.03, p=0.004) negatively impacted cell metabolism, the effects of which could be mitigated by high serum concentration (Effect=9.16, p=0.029). IDO activity was negatively impacted by high F/F ratio (Effect=-4.91, p=0.012) or the combination of high F/F ratio and high flowrate (Effect=-4.62, p=0.017). Trypsin concentration had no significant impact on cell quality. Conclusion Flowrate and F/F ratio are critical factors in CFC processing and are key considerations for process development. While MSCs were the cell types tested in this particular study, they are also likely to impact on other cell types.