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. V C 2016 AIP Publishing LLC.

Deformability-induced lift force in spiral microchannels for cell separation

“…9 Currently, only a limited number of fluorophore-conjugated antibody reagents are suitable for clinical processing 10 and the adverse effects of introducing these probes into patients are unknown, but it is generally recognised that they could potentially trigger immune and toxic responses. 11 Separation of cells based on label-free biomarkers has been recognised as a viable alternative to conventional techniques such as FACS and MACS. 12,13 Label-free biomarkers are cost-effective since there is no need to add costly antibodies to reveal cell identity markers and the number of processing steps (staining, washing) is reduced.…”

Biophysical subsets of embryonic stem cells display distinct phenotypic and morphological signatures

“…Young’s modulus was also used as a binary classifier in a previous study that established stiffness-based sorting of ESCs [ 60 ]. Tangential flow filtration was employed to separate ESCs from ESC-derived osteoblasts or fibroblasts based on Young's modulus.…”

Challenges and advances in scale-up of label-free downstream processing for allogeneic cell therapies

“…Zhang et al describe in their proof-of-principle paper successful separation of two cancer cells with different deformability using a TFF chip device, but with limited efficiency (the fraction of the more elastic cells increased from 50 to 73%). Willoughby et al reported similar results when separating pluripotent and differentiated osteoblastic cells, and pluripotent cells and fibroblasts [46,47]. A study comparing various silicon-based microfilters to separate white blood cells from red blood cells indicates that TFF is superior over NFF and weir and pillar type filters [48].…”