Design of bioreactors for mesenchymal stem cell tissue engineering

The ex vivo manufacture of functional organs and tissues for implantation can impact on therapeutic needs arising from an ageing population. The rapid expansion of the field of tissue engineering has arisen in response to this clinical need. In order for mesenchymal stem cells (MSC) to be useful clinically, sufficient numbers must be obtained. It was hypothesised that MSC could be isolated from a heterogeneous population of cells, and that hollow fibre bioreactor systems may be a scalable technology for expansion. In order to manufacture functional tissues, appropriate bioreactor devices must be developed to direct cellular differentiation. It was hypothesised that long-term ex vivo tissue gradients could be established.A substrate (Aldefluor) for aldehyde dehydrogenase (ALDH) activity was employed to label cells from human umbilical cord blood (hUCB) and cells isolated from rat, murine, porcine and equine origins. Growth of anchorage dependent cells was carried out in cuprophan hollow fibres coated with a novel recombinant protein, with and without extracapillary co-culture. Micro bioreactors were manufactured using the techniques of lithography from polydimethylsiloxane, and device biocompatibility was assessed using the anchorage dependent cell line NIH 3T3.While cells isolated from rat, murine, porcine and equine origins showed an ALDH bright population, MSC could not be purified from hUCB. Cellular attachment to cellulose based substrates coated with the recombinant protein occurred within 2 hours. Anchorage dependent cells could not be maintained in the hollow fibres. Stable concentration gradient profiles were generated experimentally in two micro-devices with differing geometries. In the second device, the concentration gradient was maximal in the region of flow stasis, and cells were found to remain viable inside the cell chamber below a flow rate of 4μL/min for 72 hours.Aldefluor substrate was not useful in the prospective isolation of MSC from a heterogeneous population. Coating of cellulose substrates with a novel recombinant protein was found to be necessary for cell attachment. Growth in the hollow fibres was found to be suboptimal compared to conventional methods. Cell chemotaxis and tissue morphogenesis could be studied using the second micro-device developed without the confounding effect of fluid shear stress