Simple Isolation of Human Bone Marrow Adipose Tissue–Derived Mesenchymal Stem/Stromal Cells

Abstract Bone marrow adipose tissue (BMAT) has garnered significant attention due to its critical roles in leukemia pathogenesis, cancer metastasis, and bone marrow failure. BMAT is a metabolically active, distinct tissue that differs from other fat depots. Marrow adipocytes, closely interacting with hematopoietic stem/progenitor cells and osteoblasts, play a pivotal role in regulating their functions. However, standardized methods for isolating and defining human BMAT (hBMAT) remain unclear. In animal models, BMAT is commonly isolated directly from the bone marrow through flushing, enzymatic digestion, or mechanical disruption. In humans, BMAT isolation often involves the adipogenic induction of bone marrow mesenchymal stem/stromal cells (BM‐MSCs) derived from bone marrow aspirates. However, this approach reflects cellular responses to chemical stimuli and may not accurately represent in vivo differentiation potential. Similarly, BMAT obtained from hip or knee replacement surgeries might not reflect basal physiological conditions due to inflammatory influences. Here, we describe a direct method for culturing BMAT from the fatty layer of bone marrow aspirates obtained from healthy transplant donors. This protocol employs centrifugation and washing steps using basic laboratory equipment, offering simple and non‐enzymatic approach. For validation, isolated cells are characterized according to the International Society for Cell & Gene Therapy (ISCT) criteria. © 2025 Wiley Periodicals LLC. Basic Protocol 1 : Isolation of human BMAT‐MSCs from the fatty layer of the bone marrow Basic Protocol 2 : Culture expansion, trypsinization, and cryopreservation of BMAT‐MSCs Support Protocol 1 : Immunophenoypic characterization of human BMAT‐MSCs by flow cytometry Support Protocol 2 : In vitro characterization of multilineage differentiation potential of human BMAT‐MSCs Support Protocol 3 : Further characterization of gene expression in human BMAT‐MSCs using qRT‐PCR