ENHANCED EXPANSION OF HUMAN BONE MARROW- AND ADIPOSE TISSUE DERIVED- MESENCHYMAL STROMAL CELLS IN AN IMPROVED ANIMAL COMPONENT-FREE CULTURE MEDIUM

Background & Aim MesenCult™-ACF-Plus (MACF-P) is an improved animal component-free (ACF) culture medium for the derivation and expansion of mesenchymal stromal cells (MSCs) from primary tissues. We characterized MSCs derived from human bone marrow (BM) and adipose (AD) tissues in MACF-P and in medium containing fetal bovine serum (FBS). Methods, Results & Conclusion Clonogenic growth was evaluated by plating BM mononuclear cells (MNCs) or AD-derived stromal cells (SCs) at low density in the Colony-Forming Unit-Fibroblast (CFU-F) assay. The proliferative potential of BM and AD-derived MSCs was measured by determining cell number at each passage (P) up to P6 (BM-MSCs) or P9 (AD-MSCs), and paired t-test was used for statistical analysis. MSCs from both tissue sources were plated at 1.5–3 × 103 cells/cm2 in each medium for long-term cell expansion. Immunosuppression of CD4+ T cells by BM-MSCs cultured in MACF-P was evaluated by co-culture of BM-MSCs with PBMCs. The PBMCs were labelled with eFluor450, activated with human CD3/CD28 T Cell Activator and then analyzed by flow cytometry for T cell proliferation after 5 days. Total CFU-F per 106 AD-SCs and per 106 BM-MNCs was comparable in MACF-P and FBS containing medium (AD-SCs: 236 ± 127 vs. 268 ± 120 [mean ± SEM; n=3]; BM-MNCs: 37 ± 7 vs. 32 ± 5 [mean ± SEM; n=6] in MACF-P and FBS-containing media, respectively). However, the average fold-expansion of AD-MSCs at each subculture over 9 passages was significantly higher in MACF-P medium (12.2 ± 0.9; mean ± SEM; n=5) than in FBS-containing medium (3.8 ± 0.3; mean ± SEM; n=5; p<0.05). Similarly, the average fold-expansion of BM-MSCs at each subculture over 6 passages was also significantly higher in MACF-P (11.6 ± 1.2; mean ± SEM; n=5) than in FBS-containing medium (3.9 ± 0.4; mean ± SEM; n=5; p<0.05). Both AD-MSCs and BM-MSCs cultured in MACF-P differentiated more robustly in vitro under the appropriate conditions into adipogenic, osteogenic and chondrogenic cells (as visualized by Oil Red O, Alizarin Red and Alcian Blue staining, respectively) compared to the same cells cultured in FBS-containing medium. Co-culture of BM-MSCs with PBMCs in MACF-P indicated that proliferation of CD4+ T cells is suppressed in a cell concentration-dependent manner when BM-MSCs were added in vitro. These data demonstrate that improved MACF-P medium supports derivation and efficient expansion of both BM-MSCs and AD-MSCs under complete ACF conditions and that BM-MSCs exhibit immunosuppressive activity in vitro. MesenCult™-ACF-Plus (MACF-P) is an improved animal component-free (ACF) culture medium for the derivation and expansion of mesenchymal stromal cells (MSCs) from primary tissues. We characterized MSCs derived from human bone marrow (BM) and adipose (AD) tissues in MACF-P and in medium containing fetal bovine serum (FBS). Clonogenic growth was evaluated by plating BM mononuclear cells (MNCs) or AD-derived stromal cells (SCs) at low density in the Colony-Forming Unit-Fibroblast (CFU-F) assay. The proliferative potential of BM and AD-derived MSCs was measured by determining cell number at each passage (P) up to P6 (BM-MSCs) or P9 (AD-MSCs), and paired t-test was used for statistical analysis. MSCs from both tissue sources were plated at 1.5–3 × 103 cells/cm2 in each medium for long-term cell expansion. Immunosuppression of CD4+ T cells by BM-MSCs cultured in MACF-P was evaluated by co-culture of BM-MSCs with PBMCs. The PBMCs were labelled with eFluor450, activated with human CD3/CD28 T Cell Activator and then analyzed by flow cytometry for T cell proliferation after 5 days.