MACSQuant^® Tyto^® Cell Sorter by Miltenyi Biotec B.V. & Co. KG

"Cord blood contains multiple stem cell populations, both hematopoietic and non-hematopoietic. We are interested in purifying lineage-negative CD34-negative cells which express CD31 as well a number of embryonic and induced pluripotent stem cell antigens. These cells are very rare, constituting ~ 0.2 – 0.3% of cord blood mononuclear cells. The cells are small with very little cytoplasm and their nuclei have a blast cell morphology. The sorting strategy is Hoechst 33342+ to gate on 2n cells, then gating on CD45 low vs lineage-negative and sorting CD31+ cells. We have sorted these cells both on the Tyto and a Becton-Dickinson Influx machine. We find the purity and yield of our sorts are comparable between the machines, with higher viability on the Tyto-sorted samples. Tyto-sorted samples are concentrated during the sorting, while Influx-sorted samples are not, and the unsorted population from Tyto runs can be resorted to increase the yield, which we have done one time, something that cannot be done on the Influx. The stability of the Tyto sort parameters is strikingly better than the Influx; the sorts are highly reproducible. Finally, the sealed sorting cartridge affords higher confidence of maintaining a sterile sort, which is crucial to our goal of developing a culture system for these cells. Other planned uses for the Tyto include sorting human hematopoietic stem cell subsets to characterize their activity, sorting peripheral blood lineages from allogeneic hematopoietic stem cell transplant patients to characterize lineage engraftment, and isolating genetically engineered lymphoid effector cell populations with the ultimate goal of use for patient cellular therapies."