Mesenchymal stem cells (MSCs) are a multipotent population of cells which reside in the bone marrow and have significant therapeutic potential due to their plasticity and easy accessibility. In vivo, MSCs have the ability to differentiate into tissues of a variety of lineages and are a significant source of trophic factors. These abilities are mediated via the complex in vivo microenvironment including local physiological conditions. An important approach to tissue regeneration using MSCs is to develop functional cellular constructs by combining MSCs with 3D scaffolds through in vitro cultivation. The success of this approach depends on the innate ability of MSCs to proliferate and differentiate in response to environmental cues that are provided by the 3D construct. To optimize this approach, the in vivo microenvironment of hMSCs must be closely monitored and mimicked in vitro.

We have developed a novel NMR compatible perfusion bioreactor that integrates cell seeding and growth in a 3D construct into a single device that has the flexibility to provide continuous monitoring of the local hydrodynamic and metabolic microenvironment over a period of 20 days under controlled physiological conditions. The bioreactor is capable of supporting long-term construct development and the non-invasive monitoring of metabolic, hydrodynamic, and cellular microenvironment under perfusion flow using both nuclear magnetic resonance (NMR) and magnetic resonance spectroscopy (MRS) imaging technologies. We were able to achieve a uniform cell distribution within the growth construct and demonstrate high-density hMSC growth for a period of 14-21 days. The reactor system also provides the flexibility to acquire spectroscopic data from various parts of the growth construct using MRS imaging. The specific voxels within the construct were targeted to study the local metabolic state. 1H NMR spectroscopy verified the viability of the construct by acquisition of choline and lactate spectral peaks from within the growth construct. Spectroscopy also verified an even distribution of hMSCs within the construct, with no spatial variation in metabolic activities. We also investigated the metabolic states under perfusion as the reactor was perfused within the magnet for a period of 24 hours. We were able to measure the apparent diffusion coefficient (ADC) within the 3D growth construct and determine the localized ADC. The ADC within the 3D growth construct was significantly lower than the surrounding media, indicating limited diffusion due to the presence of cellular membranes. The construct to surrounding media ADC ratio was 0.8 within the experimental reactors, compared to 0.97 in the control reactor. Using the above results we have discovered the baseline hydrodynamic environment within the perfused reactor as well as a baseline metabolic profile of the bioreactor growth construct.

Current and future experiments involve expanding on the aforementioned research. Recent studies have shown that hMSC are highly responsive to their hydrodynamic microenvironment and the presence of media flow regulates hMSC proliferation and progenicity. Our reactor can support both parallel flow around the construct and transverse flow across the construct, thus giving us the ability to study the differences in diffusive and convective transport of macromolecules. Using the bioreactor and 1H MRS and ADC measurements, we will study the effects of varying the flow mode has on the osteogenic differentiation of hMSCs within the 3D growth construct. Differing the flow conditions within the reactor will allow for determination of the optimal method of culturing hMSCs within a 3D construct so that they may have the most beneficial effect on growing a therapeutically useable tissue construct.