Different behaviour of fresh and cultured CD34(+) cells during immunomagnetic separation

In-vitro expansion of human cord blood (CB) cells could enhance peripheral blood recovery and ensure longterm engraftment of larger recipients in the clinical transplant setting, Enrichment of CD34(+) cells using the MiniMACS column has been evaluated for the preparation of CB CD34(+) cells before a nd after expansion culture, Repurification of CD34(+) cells after culture w ould assist accurate phenotypic and functional analysis. When fresh CB mono nuclear cells (MNC) were separated, the MACS positive (CD34(+)) fraction (9 0.1% pure) contained a mean (+/-SD, n = 5) of 93.0 +/- 8.0% of the eluted C D34(+) cells, 99.6 +/- 0.7% of the CFU-GM and all of the eluted long-term c ulture-initiating cells (LTC-IC). Cord blood CD34(+) cells were then cultur ed for 14 d with IL-3, IL-6, SCF. G-CSF and GM-CSF, each at 10 ng/ml. The t otal cell expansion was 2490 +/- 200-fold and the CD34(+) cell expansion wa s 49 +/- 17-fold. The percentage of CD34(+) cells present after expansion c ulture was 1.2 +/- 0.85%. When these cells were repurified on the MiniMACS column, the MACS positive fraction only contained 40.3 +/- 13.4% of the elu ted CD34(+) cells which was enriched for the mature CD34(+) CD38(+) subset, 24.4 +/- 8.8% of the eluted CFU-GM and 79.5 +/- 11.0% of the LTC-IC. The r emaining cells were eluted in the MACS negative fraction, in conclusion, re purification of cultured CD34(+) cells does not yield a representative popu lation and many progenitors are lost in the MACS negative fraction, This ca n give misleading phenotypic and functional data. Cell losses may be import ant in the clinical setting if cultured cells were repurified for purging.