LAUSR

We previously reported the enrichment and sorting of small CD31+ cells from human umbilical cord blood by magnetic bead depletion of lineage+ and CD34+ cells. We also reported different lineage/CD34+ depletion strategies [combining Lineage depletion and CD34+ UltraPure kits vs Diamond CD34+ kit] results in different input populations for sorting; CD31+ cells could be sorted from either. Here we report adding the missing component from the alternate lineage-depletion cocktail [anti-CD61 microbeads to Lineage depletion kit/anti-CD123 microbeads to Diamond kit] results in identical input populations, with the CD31+ target cells comprising >20% of input population, permitting rapid, high-purity sorting. We have performed sorts with Influx [B-D] and Tyto [Miltenyi] sorters, yielding 125,000 to 750,000 cells [92-98% pure] in 15 minutes. We show purified small CD31+ blast-like cells express the ESC marker alkaline phosphatase, as well as CD4, CD90 and CxCR4. We have been able to successfully isolate our CD31+ cell population from frozen cord blood samples stored in DMSO/FBS after red cell depletion. Frozen storage results in decreased MNC recovery, but CD31+ cell yields appear unaffected, further improving the enrichment of the CD31+ cell population. This is similar to what we have seen with frozen samples for CD34+ HSC isolation. The ability to use frozen samples is greatly improved by the using of Miltenyi's Tyto Running Buffer, a cGMP-compliant wash and transport solution which contains Tytonase to prevent cell clumping. Tyto Running Buffer can be used in the magnetic bead depletion steps without affecting overall yields. These findings permit rapid sorting of highly purified CD31+ cells, which can be used for gene expression profiling and cell culture.