3-D culture model is designed to mimic the in vivo microenvironment of the BM. (A) Diagram of the 3-D BM culture setup with fibronectin/collagen I surface coating to reconstruct rEnd, overlaid with BMCs mixed with Matrigel/fibronectin ECM, covered with growth medium (left panel). Cellular composition of the 3-D culture assessed after 3 weeks (right panel). Cytospins were prepared from the preculture BM and from the same samples after culture (n = 6 individually analyzed patients) and stained with May-Grunwald-Giemsa. Nuclear and cytoplasmic staining and morphology were analyzed, and cells of various lineages were counted and reported as percentage of total cellularity. Blast indicates all blasts; PMN, polymorphonuclear (bands, neutrophils, eosinophils, basophils); pre-PMN, promyelocyte, myelocyte, metamyelocyte; lymphocyte, B and T cells; NRC, nucleated red cells (proerythroblast, basophilic erythroblast, polychromatophilic erythroblast, orthochromatic erythroblast); other, macrophages, stromal cells, reticular cells, osteoclastic cells, osteoblastic cells. (B) BMCs grown in rBM recapitulate multicellularity of the BM. BMCs were grown in 3-D for the indicated number of days, and the overall view of the culture was analyzed using brightfield microscopy (original magnification ×200; see “Microscopy”). Open arrows and inset represent migrating cells; large black arrows, B-cell colonies; large white arrows, PC colonies; striped arrows, stromal cells. Representative images from 48 individually analyzed patients. (C) Proliferation of the various cellular compartment of the rBM was measured by labeling BMCs with CFSE (n = 4 individually analyzed patients) and culturing in 3-D for the indicated number of days. Cells were isolated from 3-D, stained with fluorescent tagged antibodies as indicated, and proliferation capacity of the aggregate rBM (*P = .001), CD20⁺ B cells (*P = .024, **P = .006), and CD138⁺ PCs (*P = .004) was measured by multicolor flow cytometry (inset, proliferation profile of CD138⁺CD56⁺ cells). Percentage CFSE labeled cells represents cells with average amount of retained CFSE above the unlabeled control. Error bars in panels B and D represent SEM.