Human mesenchymal stem cells (hMSCs) are readily isolated from adult donors, proliferate rapidly in culture, differentiate into various tissue types, and have great potential to develop into cellular constructs useful in a wide range of clinical applications. As highly sensitive cells to the in vivo physiological environment, hMSCs are greatly influenced by their in vitro culture environment. Perfusion bioreactor systems play a vital role in three-dimensional (3-D) construct production, and shear stress is an important biomechanical parameter in regulating hMSC construct development. In planar surfaces, shear stress induced by media flow is a known factor that regulates cellular events including cell proliferation, differentiation, and extracellular matrix (ECM) section. The effects of shear stress on 3-D hMSC construct development, however, is not fully understood because of the variations in bioreactor design and strong dependence of shear stress distribution on the spatial structure of the construct. In this study, we first evaluated the effects of flow rate on hMSC expansion in 3-D perfusion bioreactor under parallel flow mode. The hMSCs within the 3-D porous poly(ethylene terethphthalate) (PET) showed different growth, ECM expression, progenicity and osteogenic differentiation potential when cultured under the flow rates of 0.1 and 1.5 mL/min over a 20-day culture period. Specifically, a 1.4 times higher proliferation rate, higher colony forming unit-fibroblast (CFU-F) formation, and higher fibronectin and HSP-47 secretion at day 20 were observed at the flow rate of 0.1 mL/min compared to those at the flow rate of 1.5 mL/min. The higher flow rate of 1.5 mL/min, however, upregulated osteogenic differentiation at day 20 as measured by the expression of alkaline phosphatase activity and calcium deposition in the matrix after 14 days induction, consistent with those reported in literature. Mathematical modeling indicated that shear stress existed in the range of 1Χ10^-5 to 1Χ10^-4 Pa in the constructs up to a depth of 70 μm due to flow penetration in the porous constructs.

There are primarily two types of perfusion systems defined by the relative relation between the construct and media flow: transverse flow (i.e., media flow through the construct) and parallel flow (i.e., media perfuse on the surface of the construct). The results from these two types of the perfusion bioreactor systems vary significantly and no direct comparison is yet available. We are currently evaluating the effects of two operational modes on hMSC construct development by operating two chambers in transverse flow and the other two under parallel flow within the same system of four flow chambers. Preliminary results showed that parallel flow mode has higher cell and CFU-F numbers than that of the transverse flow over a 20-day culture period. Taken together, these results demonstrate that shear stress is a significant regulator for 3-D hMSC construct development and that bioreactor configuration is an important consideration for designing the perfusion bioreactor system.

References:

1. Zhao F, Chella R and Ma T. Effects of shear stress on 3-D human mesenchymal stem cell construct development in a versatile perfusion bioreactor system: experiments and hydrodynamic modeling. Biotechnology and Bioengineering, in review.

2. Zhao F, and Ma T. Perfusion bioreactor system for human mesenchymal stem cell tissue engineering: dynamic cell seeding and constructs development. Biotechnol Bioeng 2005;91:482-493.

3. Zhao F, Pathi P, Grayson WL, Xing Q, Locke BR, and Ma T. Effects of oxygen transport on 3-D human mesenchymal stem cell metabolic activity in perfusion and static cultures: experiments and mathematical model. Biotechnol Prog, 2005;21:1269-1280.