Optimization of CD34⁺ hematopoietic stem and progenitor cell isolation from human peripheral blood and evaluation of short-term culture conditions.

The recovery of viable, high-purity CD34⁺ cells from peripheral blood remains challenging for research applications. This study describes an optimisation process beginning with traditional Ficoll-based density gradient centrifugation and progressing to SepMate™ technology, combined with different magnetic separation systems and culture conditions, to establish an improved protocol for CD34⁺ cell isolation and subsequent culture for further research use. Early trials focused on cell concentration and seeding densities in stationary culture. Comparison of different density-gradient approaches indicated that the multiple centrifugation steps and prolonged processing times associated with Ficoll-based enrichment reduced CD34⁺ cell purity and viability compared with enrichment using SepMate™ tubes with Lymphoprep™. Column-based separation involving plunger-mediated elution was associated with reduced viability in the tested samples, although the effects of elution pressure could not be separated from processing duration or donor-specific variability. Despite multiple optimisation steps across enrichment, magnetic separation, and culture conditions, expansion of the cultured cells was not achieved. The methodological adjustments described here aim to improve the early handling of CD34⁺ cells from peripheral blood to provide a more reliable starting population for downstream culture studies and may facilitate the establishment of similar workflows in other laboratories.•The use of SepMate™ tubes for PBMC isolation improved cell viability and recovery compared with Ficoll-based density gradient centrifugation.•Different magnetic separation systems and culture conditions were evaluated to maintain cell viability and purity during culture.•Agitation during culture supported maintenance of viable cells; however, after seven days the total cell number remained low and did not exceed the initial cell input.