A three-dimensional (3D) bone marrow (BM) culture system may facilitate research into the molecular mechanisms involved in hematopoiesis and BM diseases. However, because >90% of BM cells are composed of non-adherent blood cells, it is difficult to organize the dispersed BM cells into 3D multicellular spheroids using conventional aggregation methods such as hanging drop, and rotary shaking culture. The objective of this study was to reproduce BM-like tissue. We reported successful formation of BM aggregates using a 3% methylcellulose (MC) medium. This medium could aggregate even non-adherent materials. In MC medium, BM cells formed tissue-like aggregates within 24 h. Although the cell density of the BM-like tissue is slightly low, sections of the organoids resembled those of intact BM tissue. Cells of the BM-like tissue were approximately 70% viable after 7 days in culture. Staining for CD68, PDGFRα, and CXCL12 indicated that the BM-like tissue contained macrophages, and mesenchymal cells including CXCL12-abundant reticular cells. These results indicated that the method using MC medium effectively reconstitutes the BM-like tissue.
Keywords: 2D, Two-dimensional; 3D, Three-dimensional; Aggregate; BM, Bone marrow; Bone marrow; CAR cell, CXCL12-abundant reticular cell; CXCL12, Chemokine (C-X-C motif) ligand 12; DMEM, Dulbecco's Modified Eagle Medium; FBS, Fetal bovine serum; HE, Hematoxylin-eosin; HSCs, Hematopoietic stem cells; MC, Methylcellulose; Methylcellulose; PBS, Phosphate buffered saline; PDGFRα, Platelet-derived growth factor receptor alpha; PFA, Paraformaldehyde; Three-dimensional culture; Tissue engineering.