(430n) Improvement of Cell Proliferation on 3d-Scaffold with a Perfusion Bioreactor

The potential of human stem cells in regenerative medicine and drug discovery depends on their high proliferative capacity, maintenance of progenicity, and plasticity. However, the promise of hSC to provide unlimited supply of cells for therapeutic applications also depends on the availability of a culture system for their expansion in 3D-scaffold. To achieve a high cell proliferation rate and subsequent differentiation of cellular constructs, a perfusion bioreactor system is needed; a static culture system can not ensure enough oxygen and nutrient delivery. In the present work we developed an 8 chamber dynamic culture system for ex vivo cell expansion on 3D-scaffold and we tested its functionality with C2C12 and satellite cells. This system was designed with 8 independent units composed of a culture chamber and a gas exchanger each. A multichannel peristaltic pump ensured medium perfusion in every unit. The gas exchanger unit was realized with a gas permeable and not porous silicon membrane and it was designed to ensure adequate oxygen concentration also at high cell density. Thanks to an accurate bioreactor design a uniform medium perfusion throughout the scaffold was obtained and it was verified with a colored tracer pulse. 3D collagen scaffolds of 7mm diameter and 3mm thickness were seeded with 5?10^5 ÷ 1?10^6 cells and introduced in the bioreactor chambers. For a deeper investigation of cell migration inside the scaffold we overlapped two scaffolds, one seeded at the top and one not seeded at the bottom and we analyzed cell number and vitality in these two parts. The cultures were carried on for 4, 7 and 10 days both in dynamic and in control static culture. After collagen digestion by collagenase the cell number was estimated and their vitality was investigated with BrdU and PI flow-cytometry analysis. Bioreactor improved cell culture condition allowing a higher cell proliferation if compared with static culture. In the upper scaffold only 50% of the cells present in collagen cultured in bioreactor were found after static culture. The percentage went down at 30% if we look at the not seeded scaffold. Cell vitality was investigated through PI flow-cytometry, looking at the live/dead cell percentage. These data showed 75% of living cells in static condition after 4 days, but the percentage rose up at 95% when the culture was carried on in our bioreactor. Interesting information was obtained also studying the cell cycle with BrdU and PI flow-cytometry analysis of dynamic culture: the percentage of proliferating cells decreased with time but it was always higher in the lower scaffold. These data confirm that a good medium perfusion can ensure adequate oxygen and nutrient concentration inside the scaffold and allow a homogeneous cell expansion. Homogeneity of cell distribution in dynamic culture was also confirmed by histological analysis. Our findings show how our multi-chamber bioreactor can be a useful tool for ex vivo cell expansion. Not only it is easy to handle and with a reduced hold up but also it ensures a uniform medium perfusion i.e. a high oxygen and nutrients concentration inside the scaffold, allowing a uniform cell proliferation.