title: A scalable label-free approach to separate human pluripotent cells from differentiated derivatives.
abstract: The broad capacity of pluripotent human embryonic stem cells (hESC) to grow and differentiate demands the development of rapid, scalable, and label-free methods to separate living cell populations for clinical and industrial applications. Here, we identify differences in cell stiffness, expressed as cell elastic modulus (CEM), for hESC versus mesenchymal progenitors, osteoblast-like derivatives, and fibroblasts using atomic force microscopy and data processing algorithms to characterize the stiffness of cell populations. Undifferentiated hESC exhibited a range of CEMs whose median was nearly three-fold lower than those of differentiated cells, information we exploited to develop a label-free separation device based on the principles of tangential flow filtration. To test the device's utility, we segregated hESC mixed with fibroblasts and hESC-mesenchymal progenitors induced to undergo osteogenic differentiation. The device permitted a throughput of 10(6)-10(7) cells per min and up to 50% removal of specific cell types per single pass. The level of enrichment and depletion of soft, pluripotent hESC in the respective channels was found to rise with increasing stiffness of the differentiating cells, suggesting CEM can serve as a major discriminator. Our results demonstrate the principle of a scalable, label-free, solution for separation of heterogeneous cell populations deriving from human pluripotent stem cells.
authors: Institute for Biological Chemistry, Biophysics and Bioengineering, School of Engineering and Physical Sciences , Heriot-Watt University , Edinburgh EH14 4AS, United Kingdom.
authors: N A
authors: NA
authors: Willoughby
authors: Institute for Chemical Sciences, School of Engineering and Physical Sciences , Heriot-Watt University , Edinburgh, United Kingdom.
authors: H
authors: 0000-0002-6778-7464
authors: H
authors: Bock
authors: Centers for Clinical Brain Sciences and Regenerative Medicine, University of Edinburgh , Edinburgh EH16 4SB, United Kingdom.
authors: M A
authors: MA
authors: Hoeve
authors: Centers for Clinical Brain Sciences and Regenerative Medicine, University of Edinburgh , Edinburgh EH16 4SB, United Kingdom.
authors: S
authors: S
authors: Pells
authors: Institute for Biological Chemistry, Biophysics and Bioengineering, School of Engineering and Physical Sciences , Heriot-Watt University , Edinburgh EH14 4AS, United Kingdom.
authors: C
authors: C
authors: Williams
authors: Institute for Biological Chemistry, Biophysics and Bioengineering, School of Engineering and Physical Sciences , Heriot-Watt University , Edinburgh EH14 4AS, United Kingdom.
authors: G
authors: G
authors: McPhee
authors: Centers for Clinical Brain Sciences and Regenerative Medicine, University of Edinburgh , Edinburgh EH16 4SB, United Kingdom.
authors: P
authors: 0000-0002-4551-7818
authors: P
authors: Freile
authors: Institute for Biological Chemistry, Biophysics and Bioengineering, School of Engineering and Physical Sciences , Heriot-Watt University , Edinburgh EH14 4AS, United Kingdom.
authors: D
authors: 0000-0002-0573-3620
authors: D
authors: Choudhury
authors: Centers for Clinical Brain Sciences and Regenerative Medicine, University of Edinburgh , Edinburgh EH16 4SB, United Kingdom.
authors: P A
authors: 0000-0003-0745-2504
authors: PA
authors: De Sousa