Many mesenchymal stem cell (MSC) based therapies have the common issue ofcells becoming trapped in the lung vasculature. Our novel enucleated cell therapy, named “Cargocytes,” also encounter this issue, though to a lesser degree due to their smaller size. Lung trapping can be especially dangerous in a therapeutic because it can cause embolisms in the lung vasculature. We therefore designed and optimized a series of protocols to further reduce the cell size of these Cargocytes and obtain minimal lung trapping after intravenous injection. By using a spinner flask for 3D culture, we show that the size of the MSCs and Cargocytes can be significantly reduced after 48 hours of culturing, as compared to cells cultured in a 2D environment. After optimizing the spinner flask culture, we then proceeded to optimize the enucleation process for 3D cells such that we could get a sufficient yield at an acceptable efficiency. We next compared the cell surface markers of 2D MSCs and 3D MSCs. Markers for MSCs remained constant even after culturing in 3D for 48 hours. Because Cargocytes are a novel platform technology, some diagnostic assays have yet to be conducted on these new cell carriers. We conducted a series of tests to look at markers of apoptosis, such as an Annexin V and multi-caspase assay. We found the profiles to be comparable to that of the parental MSCs. Finally, we engineered hTERT-MSCs with exogenous CDK4 to potentially allow cells to grow under 3D culture conditions. Although the CDK4 engineering did not promote growth of MSCs in 3D conditions, the cells were able to survive much longer. A cell cycle analysis also revealed that these newly engineered cells have slightly more cells in the G2 phase compared to parental hTERT-MSCs.