Human iPS- human derived tissues 3D vascular networks for patient-specific drug screening Monica Moya,1 Christina Tu,1 Luis F. Alonzo,1 Leslie Lock,1 and Steven C. George 1 1.University of California, Irvine, Irvine, CA, USA Introduction Developing dynamic 3D vasculature-fed patient-specific in vitro human microtissues has the potential to provide whole new opportunities for drug discovery and toxicity screening. In order to create patient-specific vascular network models, our work will expand on our previous work in developing microfluidic devices that can support a metabolically active stroma with culture medium perfused human capillaries.1 In this study we aim to develop vessel networks derived from human induced pluripotent stem cell-derived endothelial cells (iPS-EC). Using iPS-derived human cells provide the potential for patient-specific drug screening or “personalized medicine”. Materials and Methods Endothelial cells were derived from the human iPS cell line WTC11 (courtesy of Dr. Bruce Conklin) using an unpublished method provided by Dr. James Thompson (University of Wisconsin). The resulting differentiated population was purified using magnetic bead sorting for CD31, an endothelial cell surface marker. Human IPSECs were then grown in fully supplemented EGM-2 (Lonza) media prior to loading into a polydimethsiloxane microfluidic device. The device consists of 2 fluid filled microfluidic channels on either side of a 12 mm-sized diamond shaped tissue microchambers. Human iPS-ECs (2.5x106 cells/ml) or cord blood endothelial colony forming cell-derived endothelial cells (ECFC-ECs) were co-seeded into the central chamber with normal human lung fibroblast (NHLFs, 5x106 cells/ml) in a fibrin matrix. Formation of vessels was encouraged by both mechanical (pressure gradients, insterstitial flow) and chemical stimuli (hypoxia and nutrient deprivation). Formed network was visualized using fluorescent imaging of CD31 stained tissues. Results Cells remained viable under flow conditions in the microfluidic device through 10 days of culture. Vessel assembly and lumen formation were noted within a week of culture. By day 10 human iPS-ECs supported by stromal cells formed vessel networks whose area encompassed 16 ± 0.03% of the chamber. (Fig 1) Compared to control devices using primary ECFC-ECs in place of hiPS-ECs (Fig 2), vessel networks formed by hiPS-ECs encompassed less area of the chamber (ECFCEC vessel area: 25±0.07 %). Vessels formed by hiPS- ECs were also noted to be slightly larger in diameter (d= 19 ± 5µm) compared to ECFC-EC controls (d = 12 ± 4µm). 200 µm Fig. 1. Fluorescent microscopy of CD31 (green) stained vessel network at day 10 (a) depicts the ability of iPS-ECs to form lumenized (b) vessel structures under constant flow conditions. Fig. 2. Controls devices using ECFC-ECs form robust networks in microfluidic device by day 10 Discussion and Conclusions Our system is amendable to create vessel networks from human induced pluripotent endothelial cells. The potential to re-create complex multi-cellular micro-organs completely from the same genetic source represents a significant advance in patient-targeted therapy and increased efficacy. Ongoing current work is focused on introducing additional cells including cardiomyoctes derived from the same hiPS source. References 1. Moya, ML., Hsu, YH., Hughes CW., Lee A., George SC., Tissue Engineering, Part C. In Press Acknowledgments This work was supported by the NIH UH2-TR00481, NHLBI F32HL105055 (MLM) and NIH/NCI F3110765866 (LFA) Authors have nothing to disclose.
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