284 HIGH-THROUGHPUT MICROFLUIDIC TECHNOLOGIES FOR STEM CELL RESEARCH

Abstract

One of the major issues in stem cell biology is to determine the conditions that enable stem cell culture, which has been slow and laborious due to the present nature of culture systems. Our current research is designed to leverage existing robotic and fluid handling technologies with the unique fluid control and microenvironment properties of the microscale, along with the extensive expertise in stem cell research. One key advantage of microfluidic systems for stem cell research is the ability to ultra-miniaturize the cell-based assays. Swine adipose-derived stem cells (ADSCs) were cultured and differentiated into adipogenic and osteogenic cells. A fluid handling robot was used to implement the passive pumping in the microdevices, which did not require any fluid connectors. A 192-channel micro-conduit polydimethylsiloxane (PDMS) array was made using soft lithography and bonded onto a standard microplate. The fluid handling robot was programmed to load cells and change medium. The total time to change medium for 192 channels was 30 min. Swine adipose-derived stem cells were cultured using DMEM + 10% fetal bovine serum (FBS) for six days in a 100% humidified 5% CO₂ atmosphere. Two different cell concentrations were compared (1 × 10⁶ cells and 2 × 10⁶). The ADSCs were differentiated into adipogenic and osteogenic cells using specific differentiation media for the following ten days. The medium was changed every 24 h by the fluid handling robot. To assess for the differentiation, the adipogenic and osteogenic cells were stained using oil red O and alizarin red S to verify fat and calcium formation, respectively. Fatty acid accumulation was confirmed by red-stained lipid vesicles inside the adipogenic cells, and calcium formation was observed as red-stained calcium deposits around the osteogenic cells. We also determined that the concentration of 1 × 10⁶ cells (equivalent to 1500 cells per channel) gave better results than the concentration of 2 × 10⁶ cells (equivalent to 3000 cells per channel) in terms of cell morphology and differentiation parameters. The use of multifactorial directed differentiation using high-speed robotic systems, as employed in our research, will enable the examination of large matrices of culture and differentiation conditions for stem cells. Furthermore, our approach enables the use of gene expression analysis and other analytical methodologies to study the differentiation and cell function of cells cultured under essentially unlimited conditions. Using the automated microscale system in large factorial experiments allows analysis of the basic mechanisms underlying stem cell development in vitro, and ultimately in vivo.

This research was supported by the Illinois Regenerative Medicine Institute (IRMI).