[Establishment of a High-Viability Isolation, Identification, and Short-Term Culture System for Human Primary Acute...

To establish and optimize a protocol for the efficient isolation, identification, and short-term culture of primary CD34⁺ acute myeloid leukemia (AML) cells from bone marrow (BM) and peripheral blood (PB) samples of newly diagnosed AML patients, providing a stable primary cell model for basic and translational research on AML.

BM and PB samples were collected from 10 newly diagnosed AML patients. Cells were enriched using Ficoll density gradient centrifugation combined with CD34⁺ immunomagnetic bead sorting. Sorting efficiency was evaluated by flow cytometry via CD34⁺/CD117⁺ immunophenotyping, and cell morphology was observed using Wright-Giemsa staining. Cell counts and CCK-8 assays were employed to dynamically monitor the proliferation and viability of BM- and PB-derived cells over 72 hours. Furthermore, the effects of 10% versus 20% fetal bovine serum (FBS) concentrations on the proliferation of BM-derived cells were compared.

Following immunomagnetic sorting, the purity of CD34⁺ cells from both BM and PB exceeded 90% (92.7%±2.7% for BM, 91.1%±3.0% for PB, P>0.05), with a high proportion of CD117⁺ co-expression (71.9%±3.3% for BM, 72.6%±3.0% for PB, P>0.05). The cells exhibited typical primitive myeloid morphology. During the 72-hour culture period, the peak proliferation at 48 hour (approximately 4.0×10³±0.2×10³ viable cells) and metabolic activity (OD₄₅₀ value of 1.59±0.07) of BM-derived cells were significantly higher than those of PB-derived cells (3.2×10³±0.1×10³, 1.14±0.08, both P<0.01), with superior morphological stability in later stages. No statistically significant difference was observed between 10% and 20% FBS regarding short-term cell proliferation, although 20% FBS showed a slight advantage in maintaining cell morphology.

This study successfully established a stable and efficient system for the isolation and short-term culture of human primary CD34⁺ AML cells. This system is applicable to both BM and PB samples. The results demonstrate that BM-derived cells possess stronger in vitro proliferative capacity and that 10% FBS is sufficient for their short-term culture requirements.