Enhanced Enrichment and Purification of Blood-Derived T-Cells Using a Novel Hydrogel Technology
Authors: Armstead Andrea , Kevlahan Sean , Reil Darcy , Bortell Rita , Brehm Michael 
Published: October 2019

Abstract
Introduction: The rapid growth of cell-based immunotherapy research in academic and industry laboratories has highlighted a clear need for an optimal method to isolate specific immune cell populations from heterogeneous samples with high viability, purity and recovery efficiency. This is a particular concern in the area of CAR T-cell immunotherapy research, since isolated cells must be both viable and free of any magnetic bead labels for clinical use (N Engl J Med 2014; 371:1507-17). Herein, we report a novel approach for isolating desired T-cell populations from heterogeneous samples, including peripheral and umbilical cord blood, using a biologically-friendly hydrogel technology (QuickGel™) with an interior magnetic bead carrier (MagCloudz™). MagCloudz™ have been engineered with an exterior streptavidin binding surface, which readily allows for target cell isolation using a biotinylated antibody directed against the cellular marker of interest.
Methods: Target cells were isolated from human samples and analyzed for marker expression and viability as follows. CD3+ T-cells were first labeled with biotinylated antibody and then bound to the MagCloudz™ streptavidin during a short incubation step. These target CD3+ T-cells were then separated from the undesired cell populations using a magnetic stand. Any cells non-specifically adhered to the MagCloudz™ were removed during a wash step and then the CD3+ T-cells were released from the MagCloudz™ using a specially designed buffer, which dissolved the QuickGel™ and effectively de-coupled the T-cells from the magnetic bead carriers. The magnetic beads were then removed using the magnetic stand. CD3/CD45 expression on isolated T-cells were determined by flow cytometry. Viability of recovered cells was determined by 7-AAD and Annexin-V staining.
Results: We demonstrate successful CD3+/CD45+ T-cell enrichment from both peripheral and umbilical cord vein blood samples. In peripheral blood, flow cytometry analysis indicated that CD3+/CD45+ T-cell populations were enriched 1.8-fold from 56% in the starting population to 98.3% CD3+/CD45+ in the isolated T-cell population, with recovery >80% and viability >90%. In umbilical cord blood, CD3+/CD45+ T-cells were enriched 4.8-fold from 17.5% in the starting population to 83.7% CD3+/CD45+ in the isolated T-cell population. Viability of the recovered T-cells was 96%, making them ideal for use in further functional studies (ongoing).
Conclusions: These data demonstrate that the novel MagCloudz™ with QuickGel™ approach described here has the potential to drastically improve the enrichment, purity and viability of isolated of immune cell populations from whole blood. We expect that this approach will reduce the cost and time of bio-processing for immunotherapy research and provide a means to deliver magnetic label-free cells for clinical applications such as CAR T-Cell therapy (N Engl J Med 2014; 371:1507-17).

Disclosures
Brehm: The Jackson Laboratory: Consultancy.