289 STEM CELL CULTURE AND DIFFERENTIATION IN MICROFLUIDIC DEVICES

Abstract

Advancements in micro and nanotechnology have allowed scientists a powerful platform to study biological systems. Microfluidics is one area of advancement with great promise. Microfluidics deals with the behaviour, specific control, and manipulation of microliter and nanoliter volumes of fluid. The small-scale design of these microfluidic devices permits laminar flow, characterised as parallel streams flowing without disruption between currents. With the introduction of micro-technology and microfluidic platforms for cell culture, stem cell research can be put into a new context. Inside microfluidics, microenvironments can be more precisely controlled and they provide a more in vivo-like environment for the cells to grow and hence can serve as a better way of culturing the cells. In the current study, we examined the influence of microfluidic devices on the development of stem cells. Adipose-derived stem cells (ADSC) were isolated from pigs and seeded in a microfluidic device to differentiate toward adipogenic, osteogenic, and chondrogenic lineages using specific differentiation-promoting media (Monaco et al. 2009 Open Tissue Eng. Regen. Med. J. 2, 20–33). Five thousand cells were seeded per channel at a density of 5 000 000 cells mL^–1. The microchannel dimensions were 5 mm long, 1 mm wid, and 200 µm deep. Cells were maintained for 14 days and then stained with respective staining dyes: Oil Red O for adipogenesis, Alizarin Red for osteogenesis, and Toluidine Blue for chondrogenesis. Cells differentiated towards adipogenic lineage contained small lipid droplets, which stained red with Oil Red O stain; during osteogenic differentiation, the cells formed large nodules and stained positive for the presence of calcium; and the chondriogenic differentiating cells showed the presence of proteoglycans (blue) when stained with Toluidine Blue. We seeded ADSC in 5 channels for each differentiation lineage, and all channels gave positive staining results. We conclude that microfluidic channels support proliferation and differentiation of ADSC. This system uses small amounts of culture medium, experiments with different culture compositions can be efficiently performed, and culture manipulations can be automated using fluid-handling robotics. Because microfluidics can deal with small number of cells, the characteristics of cellular structure and function and the microenvironment of the stem cells can be understood in a more precise manner. The miniaturization of cell culture platforms allows the observation of cellular behaviour at the scale found in living systems.