A Reliable Method for Thawing Primary AML and CMML Mononuclear Cells to Preserve Viability and Function

Human mononuclear cells derived from peripheral blood and bone marrow are valuable resources for the study of hematological malignancies, including acute myeloid leukemia (AML) and chronic myelomonocytic leukemia (CMML). Cryopreservation enables long-term storage of patient samples for downstream assays; while thawing protocols have been described, subsequent recovery of viable cells after thawing can be challenging, particularly for fragile blast and monocyte populations. Here, we describe a reliable protocol for thawing cryopreserved AML and CMML mononuclear cells designed to preserve post-thaw viability, recovery, and functional integrity. The method incorporates controlled dilution of cells out of cryoprotectant with anticoagulant-supplemented thaw buffer, DNase I treatment, and gentle resuspension steps. Using this approach, post-thaw viability consistently exceeded 80% with a mean recovery of 55.6% across samples. Recovered cells retained functional capacity, as demonstrated by colony-forming assays, and maintained immunophenotypic characteristics by flow cytometry. This protocol provides a robust and reproducible method for the recovery of cryopreserved AML and CMML mononuclear cells and may be broadly applicable to other fragile or monocyte-rich patient-derived hematopoietic samples. Key features • Reliable thawing protocol that preserves viability and recovery of cryopreserved AML and CMML mononuclear cells. • Controlled dilution with anticoagulant supplementation, DNase I treatment, and gentle resuspension minimizes cell aggregation of fragile blast and monocyte populations. • Maintains cell functional capacity and immunophenotypic characteristics for downstream assays. • Applicable to cryopreserved patient-derived mononuclear cells prone to aggregation, low recovery, or poor viability with conventional thawing.