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

Abstract

In‐vitro expansion of human cord blood (CB) cells could enhance peripheral blood recovery and ensure long‐term 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 and after expansion culture. Repurification of CD34⁺ cells after culture would assist accurate phenotypic and functional analysis. When fresh CB mononuclear cells (MNC) were separated, the MACS positive (CD34⁺) fraction (90.1% pure) contained a mean (± SD, n = 5) of 93.0 ± 8.0% of the eluted CD34⁺ cells, 99.6 ± 0.7% of the CFU‐GM and all of the eluted long‐term culture‐initiating cells (LTC‐IC). Cord blood CD34⁺ cells were then cultured for 14 d with IL‐3, IL‐6, SCF, G‐CSF and GM‐CSF, each at 10 ng/ml. The total cell expansion was 2490 ± 200‐fold and the CD34⁺ cell expansion was 49 ± 17‐fold. The percentage of CD34⁺ cells present after expansion culture 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 eluted 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 remaining cells were eluted in the MACS negative fraction. In conclusion, repurification of cultured CD34⁺ cells does not yield a representative population and many progenitors are lost in the MACS negative fraction. This can give misleading phenotypic and functional data. Cell losses may be important in the clinical setting if cultured cells were repurified for purging.