rBM maintains the architecture of in vivo BM. (A) BMCs were grown in rBM for 21 days and stained with DAPI (pseudocolored red) to mark nuclei. The stratification of the 3-D culture was assessed by confocal microscopy as a 3-D reconstruction from the confocal stack (original magnification 100×). Representative images from BM of 48 individually analyzed patient samples. (B) BM aspirated particle grown in rBM for 21 days and stained with DAPI (pseudocolored red) to mark nuclei. The stratification of the 3-D culture was assessed by confocal microscopy as a 3-D reconstruction from the confocal stack (original magnification 100×). Representative image from analysis of aspirated particles from 28 individually analyzed patients. (C) BM stromal cells redistribute to rEnd. The rBM layer was removed from 3-D culture, and the cells at rEnd were stained with markers to identify stromal components observed at rEnd (adipocytes, Oil Red; fibroblasts, fibronectin and phallodin; osteoblasts, alkaline phosphatase [ALP] and Alizarin Red [inset]; osteoclasts, tartarate-resistant acidic phosphatase [TRAP]). Brightfield microscopy (original magnification 200×). Representative images from 7 individually analyzed patients; see “Immunohistochemistry.” (D) BMCs were grown for 14 days and the percentage of CD34⁺ HPCs, B cells, or PCs in each layer (black represents fraction in bottom layer; gray, fraction in middle; white, fraction in top layer) of the 3-D culture was calculated based on the immunohistochemical staining for CD34, CD20, or CD138 (n = 4 individually analyzed patients). Error bars represent SEM. For complete image acquisition information, see “Microscopy.”