March 15, 2016 (Vol. 36, No. 6)

Andrew Ball Ph.D. vice president of research and development Quad Technologies

New Method for High-Efficiency Capture and Label-Free Release of High-Purity Target Cell Populations

Cell-based therapeutics, such as CAR-T cancer therapies, are poised to transform 21st century medical care. These innovative medicines require highly specific isolation and purification of large numbers of viable, functional cells, a process that is often technically complex and cost-prohibitive. Consequently, demand is growing for novel approaches to cell isolation that will provide more cost-effective and scalable cell separation workflows that simply and efficiently isolate cell populations with a high degree of purity and viability.

Currently, if an end user wants to isolate or purify a population of cells defined by the presence of two or more specific markers, there are two options available, FACS or MACS. FACS platforms have limited sample throughput, making processing clinical-scale (typically tens of millions of cells per single infusion for autologous therapies; billions of cells per week for allogeneic cell manufacturing) sample sizes unfeasible, operating pressures that can lead to loss of cell function or viability, and safety concerns regarding contamination due to the sorting of aerosolized samples.

MACS systems are capable of processing much larger numbers of cells than FACS, however current MACS approaches leave large numbers of paramagnetic particles attached to cells, which is highly undesirable for cell-based therapeutics. Preclinical applications require cells to be free from contaminating particles while retaining high viability. However, minimizing the number of attached beads or, more ideally, removing them completely represents a formidable obstacle, as most magnetic-bead-based products lack the ability to readily release bound cells from capture molecules in a manner that does not alter the viability and phenotype of the isolated cells.

The next generation of cell sorting devices must have (1) clinical-scale sorting, (2) capability to isolate highly pure, uncontaminated, and viable cells, (3) capability to process native biological fluids, (4) capability to process diverse cell types, (5) capabilities for multiplexed sorting, (6) simple operating procedures, (7) reduced biohazard risk, and (8) reduced cost.

The Cell Separation Challenge

The roadblocks to effective cell separation have a significant impact on researchers in cell therapy and immunotherapy, and can hinder advancements in regenerative medicine and therapeutic development for diseases such as cancer and diabetes.

The key objective of the cell separation workflow is to obtain a highly pure and viable population of cells for downstream applications and analysis. However, most biological purification protocols lack the ability to readily release target cells from capture molecules in a manner that does not alter the viability and phenotype of the isolated cells. For instance, when isolated using conventional magnetic separation methods, the target cells may remain magnetically labeled, which results in modifications to cellular functionality and morphology and has a detrimental effect on the ability to successfully culture the cell for downstream use.

The complete removal of magnetic beads from target cells is therefore critical, especially for cell therapy applications that have strict FDA requirements for magnetic bead removal prior to therapeutic use.

Achieving High Efficiency, Purity, and Viability

To address these limitations, Quad Technologies developed QuickGel™, a unique hydrogel that can be easily functionalized with cell capture agents, such as antibodies, and is rapidly dissolvable by simply switching to a cell-friendly release buffer. This technology dramatically increases cell viability compared to conventional cell separation products and eliminates retained cell capture particles from isolated cell populations. One of the most valuable benefits of QuickGel is that the dissolution process does not affect cell phenotype or viability, which makes the isolated cells more suitable for downstream analysis and applications compared to conventional isolation technologies.

The MagCloudz™ Streptavidin Cell Separation Kit, featuring QuickGel technology, has been designed for high-efficiency capture and label-free release of high purity target cell populations from within complex biological samples. The kits overcome common cell isolation challenges with a unique magnetic separation and dissociation process, leaving isolated target cells completely label-free for scalable downstream culture and use (Figure 1).

The kit supports isolation of approximately one billion cells and can be used with any biotinylated antibody of choice allowing end user flexibility for any target cell type. The protocol takes advantage of the high affinity of streptavidin-biotin interactions coupled with the MagCloudz platform, offering high biocompatibility, low nonspecific binding, and, most importantly, high viability of magnetic label-free recovered target cells.

In order to perform cell isolation and recovery, MagCloudz Streptavidin is added to a cell sample containing biotinylated ligands. During a short incubation period, the biotinylated antibodies will bind to the MagCloudz Streptavidin forming a complex. The cell complex is then easily separated from the sample using the Q-Mag magnetic stand, also available from Quad Technologies.

The final stage is the addition of a cell release buffer that disassociates the MagCloudz cell complex resulting in a high-yield recovery of the target cells from the initial sample. The kits provide a simple cell separation workflow that delivers significant time savings over conventional methods.

Figure 1. Comparison of conventional magnetic and MagCloudz cell separation approaches

Case Study

Quad Technologies partnered with the University of Massachusetts Medical School to evaluate the performance of the MagCloudz Streptavidin cell separation kit. At UMass, the use of T cells in humanized mouse models helps to answer basic questions about human disease biology, and  the goal of the study was to isolate and purify CD3+ T cells from human umbilical cord blood (HUCB) for subsequent use in in vivo humanized mouse models. The key parameters that the kits were evaluated on were improvements in process time efficiency, improvements in graftment rates, and success in mouse models with healthier, magnetic-label-free cells and a comparable performance to conventional magnetic separation technologies.

The results in Figure 2 demonstrate the enrichment, purity, and viability of CD3+ T cell populations isolated from HUCB using the MagCloudz kit with biotinylated-anti-CD3 antibody (clone UCHT1, eBioscience). Recovered T cell populations were highly pure (>95%), highly viable (>94%), and magnetic-label-free, making them ideal for further functional studies.

Downstream assays indicated that T cells isolated from peripheral blood with MagCloudz had high viability and proliferative capacity when stimulated with a standard T cell expansion kit. A faster overall process time was also achieved. The study validated the ability of the MagCloudz solution to overcome the challenges associated with conventional cell separation approaches, delivering high-quality cells on a scalable and easy-to-use platform.

The benefits of MagCloudz will enable cell separation and cell therapy researches to develop breakthrough therapies for the clinic by overcoming the current challenges associated with cell separation techniques.       

Figure 2. Representative flow cytometry dot plots for T-cell enrichment and purification from peripheral blood. (A) The forward- and side-scatter profiles of the initial HUCB population; (B) 12.1% CD3+/CD45+ T cells (target cell population) were present in the initial HUCB sample; (C) 98% T cell uptake as indicated by the low percentage of T cells remaining in the unbound cell fraction following MagCloudz Streptavidin binding; (D) forward- and side-scatter profiles of the recovered CD3+ T cell population; (E) high-purity T cells were recovered from the MagCloudz Streptavidin, enriched from 12.1% in the initial population to 95.9% in the final isolated/released population; and (F) high viability (94.5%) of the recovered T cells, indicated by the absence of annexin-V and 7-AAD staining in the bottom left quadrant of the plot. A total of 51% of the target T-cell population was recovered as magnetic-label-free T cells from the MagCloudz Streptavidin in this assay.

Andrew Ball, Ph.D. (aball@quadtechnolo?, is vice president of research and development at Quad Technologies.

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