Background

Human primary mononuclear cells isolated from peripheral blood and bone marrow are valuable in the study of cellular processes and disease mechanisms in hematological malignancies, including acute myeloid leukemia (AML) and chronic myelomonocytic leukemia (CMML). Because access to fresh patient material is limited and experiments cannot always be performed immediately following sample collection, cryopreservation is routinely used to enable long-term storage. However, recovery of viable cells can be challenging, since thawing exposes cells to several concurrent stressors, including dimethyl sulfoxide (DMSO) toxicity at room temperature, osmotic stress leading to membrane damage and extracellular DNA release, mechanical stress from vigorous pipetting, and metabolic stress associated with increased reactive oxygen species.

Certain molecular subtypes of AML, including mutant IDH1 and IDH2, and acute promyelocytic leukemia have been noted to be particularly difficult to recover after cryopreservation. Several protocols for thawing AML cells have been described, most commonly involving rapid thawing in a 37 °C water bath followed by washing steps in serum-containing media, but issues with low viability, poor recovery, and material loss persist [1–4].

To address these challenges, here we describe a reliable step-by-step protocol for thawing cryopreserved mononuclear cells that incorporates controlled dilution of cells out of cryoprotectant to reduce osmotic stress, DNase I treatment, and gentle resuspension in an anticoagulant-supplemented thaw buffer to limit cell aggregation during recovery. These steps are designed to preserve viability, facilitate recovery, reduce sample variability, and maintain the physiological integrity of AML and CMML cells for downstream applications. These include flow cytometry–based characterization of surface markers and cytokine profiling, as well as functional studies such as colony-forming assays, phagocytosis, macrophage differentiation, antigen processing and presentation, oxidative burst, and drug-response assays. We have found our method to be particularly well-suited for CMML samples, which are typically enriched for monocytes that are prone to aggregation during thawing and can be lost during washing steps. Using this method, we consistently achieved post-thaw viability exceeding 80% with a mean recovery of 55.6% across a range of total number of cells per ampoule of cryopreserved sample, with preserved colony-forming capacity and immunophenotypic characteristics.

We routinely apply this protocol for the recovery of cryopreserved AML and CMML mononuclear cells and anticipate it may be useful for other fragile or monocyte-rich primary hematopoietic patient-derived samples.