Aptamer-Based Traceless Multiplexed Cell Isolation Systems

In both biomedical research and clinical cell therapy manufacturing, there is a need for cell isolation systems that recover purified cells in the absence of any selection agent. Reported traceless cell isolation methods using engineered antigen-binding fragments or aptamers have been limited to processing a single cell type at a time. There remains an unmet need for cell isolation processes that rapidly sort multiple target cell types. Here, we utilized two aptamers along with their designated complementary strands (reversal agents) to tracelessly isolate two cell types from a mixed cell population with one aptamer-labeling step and two sequential cell elution steps with reversal agents. We engineered a CD71-binding aptamer (rvCD71apt) and a reversal agent pair to be used simultaneously with our previously reported traceless purification approach using the CD8 aptamer (rvCD8apt) and its reversal agent. We verified the compatibility of the two aptamer displacement mechanisms by flow cytometry and the feasibility of incorporating rvCD71apt with a magnetic solid state. We then combined rvCD71apt with rvCD8apt to isolate activated CD4+T cells and resting CD8+cells by eluting these target cells into separate fractions with orthogonal strand displacements. This is the first demonstration of isolating different cell types using two aptamers and reversal agents at the same time. Potentially, different or more aptamers can be included in this traceless multiplexed isolation system for diverse applications with a shortened operation time and a lower production cost. © 2022 American Chemical Society. All rights reserved.