Label-free and high-throughput removal of residual undifferentiated cells from iPSC-derived spinal-cord progenitor cells

Spinal cord progenitor cells (SCPCs) have the capability to differentiate into multiple cell lineages of a central nervous system. The transplantation of SCPCs derived from human induced pluripotent stem cells (iPSCs) has beneficial effects on treating spinal cord injury (SCI). However, the presence of residual undifferentiated iPSCs amongst their differentiated progeny poses a high risk as it can result in the development of teratomas or other types of tumors post-transplantation. Despite the need to remove these residual undifferentiated iPSCs, there is a severe lack of surface markers that can identify them for subsequent removal. In this study, iPSCs were differentiated into SCPCs through a 10-day differentiation process. By profiling the size of SCPCs, we found that the large-sized group contains significantly more cells expressing pluripotent markers. A sized-based, label-free separation using an inertial microfluidic-based device was exploited to remove tumor-risk cells. The device takes advantage of label-free, non-contact, and high throughput (i.e., > 3 million cells per minute) without affecting cell viability and functions. The sorted cells were verified with immunofluorescence staining, flow cytometry analysis, and colony culture assay. Essentially, our technology has demonstrated its capabilities to reduce the percentage of tumor-risk cells up to 98.07 %. Our technology has great potential for the ‘downstream processing’ of cell manufacturing workflow, contributing to better quality and safety of the transplanted cells.