High‐sensitivity flow cytometers have been developed for multi‐parameter characterization of single extracellular vesicles (EVs), but performance varies among instruments and calibration methods. Here we compare the characterization of identical (split) EV samples derived from human colorectal cancer (DiFi) cells by three high‐sensitivity flow cytometers, two commercial instruments, CytoFLEX/CellStream, and a custom single‐molecule flow cytometer (SMFC). DiFi EVs were stained with the membrane dye di‐8‐ANEPPS and with PE‐conjugated anti‐EGFR or anti‐tetraspanin (CD9/CD63/CD81) antibodies for estimation of EV size and surface protein copy numbers. The limits of detection (LODs) for immunofluorescence and vesicle size based on calibration using cross‐calibrated, hard‐dyed beads were ∼10 PE/∼80 nm EV diameter for CytoFLEX and ∼10 PEs/∼67 nm for CellStream. For the SMFC, the LOD for immunofluorescence was 1 PE and ≤ 35 nm for size. The population of EVs detected by each system (di‐8‐ANEPPS⁺/PE⁺ particles) differed widely depending on the LOD of the system; for example, CellStream/CytoFLEX detected only 5.7% and 1.5% of the tetraspanin‐labelled EVs detected by SMFC, respectively, and median EV diameter and antibody copy numbers were much larger for CellStream/CytoFLEX than for SMFC as measured and validated using super‐resolution/single‐molecule TIRF microscopy. To obtain a dataset representing a common EV population analysed by all three platforms, we filtered out SMFC and CellStream measurements for EVs below the CytoFLEX LODs as determined by bead calibration (10 PE/80 nm). The inter‐platform agreement using this filtered dataset was significantly better than for the unfiltered dataset, but even better concordance between results was obtained by applying higher cutoffs (21 PE/120 nm) determined by threshold analysis using the SMFC data. The results demonstrate the impact of specifying LODs to define the EV population analysed on inter‐instrument reproducibility in EV flow cytometry studies, and the utility of threshold analysis of SMFC data for providing semi‐quantitative LOD values for other flow cytometers.

The presence of iron in circulating monocytes is well known as they play essential roles in iron recycling. Also, the storage of this metal as well as its incorrect uptake and/or release are important data to diagnose different pathologies. It has been demonstrated that iron storage in human blood cells can be measured through their magnetic behavior with high accuracy; however, the magnetic characteristics of monocytes have not been reported so far to the best of our knowledge. Therefore, in this work, we report, for the first time, the physical and magnetic properties of human monocytes, along with plasma platelets, oxyhemoglobin red blood cells (oxyHb‐RBCs), and methemoglobin red blood cells (metHb‐RBCs). The different cell populations were separated by Ficoll‐density gradient centrifugation, followed by a flow sorting step to isolate monocytes from peripheral blood mononuclear cells. The different fractions were analyzed by Coulter Counter (for determining the size distribution and concentration) and the sorted monocytes were qualitatively analyzed on ImageStream, a state‐of‐the‐art imaging cytometer. The analysis of the Coulter Counter and ImageStream data suggests that although there exists contamination in the monocyte fraction, the integrity of the sorted monocytes appears to be intact and the concentration was high enough to precisely measure their magnetic velocity by Cell Tracking Velocimetry. Surprisingly, monocytes reported the highest magnetic mobility from the four fractions under analysis, with an average magnetic velocity 7.8 times higher than MetHb‐RBCs, which is the only type of cells with positive magnetic velocities. This value is equivalent to a susceptibility 2.5 times higher than the value reported by fresh MetHb‐RBCs. It should be noted that this is the first study that reports that a subpopulation of human monocytes is much more magnetic than MetHb‐RBCs, opening the door to the possible isolation of human monocytes by label‐free magnetic techniques. Further, it is suggested that these magnetic monocytes could “contaminate” positively selected, immunomagnetically labeled blood cells (i.e., during a process using magnetically conjugated antibodies targeting cells, such as CD34 positive cells). Conversely, these magnetic monocytes could be inadvertently removed from a desired blood population when one is using a negative magnetic isolation technique to target cells for removal. © 2019 International Society for Advancement of Cytometry

The prediction of the cell yield in large-scale bioreactor culture is an important factor for various cell therapy bioprocess operations to ensure consistency in cell quality and efficient use of resources. However, the shear sensitivity of cells used in cell therapy manufacturing can make such predictions difficult, particularly in large-scale suspension cultures that have significant stresses without representative scale down models. The PBS Vertical-Wheel (VW) bioreactors have been demonstrated to provide a homogeneous hydrodynamic environment with low shear for cell culture at various scales (0.1–80 L) and is thereby employed for various shear-sensitive cells. In this study, the oxygen transfer rate for surface aeration for three large-scale VW bioreactors was measured along with the specific oxygen uptake rate (sOUR) of iPSCs cultured in the bioreactors. The oxygen mass transfer coefficient was measured in PBS-3/15/80 L bioreactors at different agitation rates, headspace gas flowrates, and working volumes using the static gassing-out method. The sOUR of iPSCs was measured using the dynamic method in the PBS-0.1 L Mini with a custom DO probe configuration. The results from both experiments were combined to calculate the theoretical maximum cell density before oxygen limitation across VW bioreactors at 2 L/3 L/10 L/15 L/50 L/80 L working volumes at a different agitation speed and aeration rate.