(443b) Effects of Macroscopic Flow On hMSCs 3-D Construct Development in Perfusion Bioreactor

Human mesenchymal stem cells (hMSCs) in three-dimensional (3D) constructs are significantly influenced by culture conditions, and perfusion bioreactor is important in regulating construct microenvironment and in providing controlled physiochemical and biomechanical environments. While both transverse and parallel flow have been used in bioreactor operation, the effects of macroscopic flow on construct microenvironment and its influence on cell behaviors and construct development have not been compared. The objective of this study is to determine the construct microenvironments under various macroscopic flows and to evaluate their subsequent effects on 3-D hMSC construct development. Three different flow regimens, including the parallel, transverse, and combination (transverse mode for 10 days and parallel mode for 10 days) at 0.1 ml/min, are produced in the in-house perfusion bioreactor system, and the effects of macroscopic flow on hMSCs' proliferation, secretion of growth factors and ECM, and osteogenic differentiation are analyzed. From the DNA result, the combination pattern maintained the higher cell density within the culture period. Interestingly, the transverse pattern had about 2.7 times higher cell numbers than ones in the parallel at day 10, while cell numbers was about 2.2 times higher in the parallel mode compared with the transverse mode at day 20. Transverse flow was also found to enhance hMSC osteogenic differentiation, which is attributed to the combined effects of shear stress stimulation and depletion of mitogenic growth factors. The effects of flow regimens on growth factor and ECM secretions are being analyzed and quantified using Western blot and ELISA, whereas proliferation and differentiation are being determined by CFU-F and qPCR. Together, the results highlight the significant impact of macroscopic flow on construct microenvironment and its influence on cell behaviors, and demonstrate the importance of modifying flow regimens to modulate construct microenvironment.